Biallelic markers derived from genomic regions carrying genes involved in arachidonic acid metabolism

ABSTRACT

The invention provides polynucleotides including biallelic markers derived from genes involved in arachidonic acid metabolism and from genomic regions flanking those genes. Primers hybridizing to regions flanking these biallelic markers are also provided. This invention also provides polynucleotides and methods suitable for genotyping a nucleic acid containing sample for one or more biallelic markers of the invention. Further, the invention provides methods to detect a statistical correlation between a biallelic marker allele and a phenotype and/or between a biallelic marker haplotype and a phenotype.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/641,638, filed Aug. 16, 2000; which is a continuation-in-part ofboth U.S. patent application Ser. No. 09/502,330, filed Feb. 11, 2000,and International Patent Application No. PCT/IB00/00184, filed Feb. 11,2000, which are continuations-in-part of U.S. patent application Ser.No. 09/275,267, filed Mar. 23, 1999. U.S. patent application Ser. No.09/502,330 and International Patent Application No. PCT/IB00/00184 arecontinuations-in-part of U.S. patent application Ser. No. 09/275,267,filed Mar. 23, 1999, and claim priority to U.S. Provisional PatentApplication Serial No. 60/133,200, filed May 7, 1999, and U.S.Provisional Patent Application Serial No. 60/119,917, filed Feb. 12,1999. Each of the above applications are hereby incorporated herein intheir entirety including any sequence lists, figures, tables, ordrawings.

FIELD OF THE INVENTION

[0002] The present invention is in the field of pharmacogenomics, and isprimarily directed to biallelic markers that are located in or in thevicinity of genes, which have an impact on arachidonic acid metabolismand the uses of these markers. The present invention encompasses methodsof establishing associations between these markers and diseasesinvolving arachidonic acid metabolism such as inflammatory diseases aswell as associations between these markers and treatment response todrugs acting on arachidonic acid metabolism. The present invention alsoprovides means to determine the genetic predisposition of individuals tosuch diseases and means to predict responses to such drugs.

BACKGROUND OF THE INVENTION

[0003] The metabolites of arachidonic acid and related fatty acids,collectively termed eicosanoids, exhibit a wide range of biologicalactivities affecting virtually every organ system in mammals.Eicosanoids are among the most important chemical mediators andmodulators of the inflammatory reaction and contribute to a number ofphysiological and pathological processes (See Hardman J. G., Goodman,Gilman A., Limbird L. E.; Goodman & Gilman's The Pharmacological Basisof Therapeutics, 9^(th) edition, McGraw-Hill, N.Y., 1996).

[0004] Physiology, Pathophysiology and Pharmacological Importance of theEicosanoids

[0005] The eicosanoids are extremely prevalent and have been detected inalmost every tissue and body fluid. These lipids contribute to a numberof physiological and pathological processes including inflammation,smooth muscle tone, hemostasis, thrombosis, parturition andgastrointestinal secretion. Once synthesized in response to a stimulus,the eicosanoids are not stored to any significant extent but arereleased immediately and act locally. After they act, they are quicklymetabolized by local enzymes to inactive forms. Accordingly, theeicosanoids are categorized as autocrine agents or local hormones. Theyalter the activities of the cells in which they are synthesized and ofadjoining cells. The nature of these effects may vary from one type ofcell to another, in contrast with the more uniform actions of globalhormones such as insulin, for example. Therefore, the eicosanoids, aslocal chemical messengers, exert a wide variety of effects in virtuallyevery tissue and organ system.

[0006] The principal eicosanoids are the prostaglandins (PG), thethromboxanes (TX) and the leukotrienes (LT), though other derivatives ofarachidonate, for example lipoxins, are also produced. They fall intodifferent classes designated by letters and the main classes are furthersubdivided and designated by numbers.

[0007] Inflammatory and Immune Responses

[0008] Eicosanoids are lipid mediators of inflammation and play acentral, often synergistic, role in numerous aspects of inflammatoryresponses and host defense. Prostaglandins and leukotrienes are releasedby a host of mechanical, thermal, chemical, bacterial, and otherinsults, and they contribute importantly to the genesis of the signs andsymptoms of inflammation. The ability to mount an inflammatory responseis essential for survival in the face of environmental pathogens andinjury, although in some situations and diseases the inflammatoryresponse may be exaggerated and sustained for no apparent beneficialreason. This is the case in numerous chronic inflammatory diseases andallergic inflammation. Acute allergic inflammation is characterized byincreased blood flow, extravasation of plasma and recruitment ofleukocytes. These events are triggered by locally released inflammatorymediators including eicosanoids and more particularly leukotrienes. Theleukotrienes generally have powerful effects on vascular permeabilityand the leukotriene LTB₄ is a potent chemoattractant for leukocytes andpromotes exudation of plasma. The prostaglandins PGE₂ and PGI₂ markedlyenhance edema formation and leukocyte infiltration in the inflamedregion. Moreover, they potentiate the pain-producing activity ofbradykinin.

[0009] The participation of arachidonic acid (AA) metabolism ininflammatory diseases such as rheumatoid arthritis, asthma and acuteallergy is well established. Prostaglandins have been involved ininflammation, pain and fever. Pathological actions of leukotrienes arebest understood in terms of their roles in immediate hypersensitivityand asthma. Lipoxygenases, e.g., 5-lipoxygenase (5-LO), 12-lipoxygenase(12-LO), 15-lipoxygenase A (15-LOA), and 15-lipoxygenase B (15-LOB),have been implicated in the pathogenesis of a variety of inflammatoryconditions such as psoriasis and arthritis.

[0010] Cardiovascular System

[0011] The prostaglandins PGEs, PGF₂ and PGD₂ cause both vasodilationand vasoconstriction. Responses vary with concentration and vascularbed. Systemic blood pressure generally falls in response PGEs, and bloodflow to most organs, including the heart, is increased. These effectsare particularly striking in some hypertensive patients. Cardiac outputis generally increased by prostaglandins of the E and F series. Theimportance of these vascular actions is emphasized by the participationof PGI₂ and PGE₂ in the hypotension associated with septic shock. Theprostaglandins also have been implicated in the maintenance of patencyof the ductus arteriosus. Thromboxane synthase (TXA2), also known asCYP5, is a potent vasoconstrictor. Leukotriene C₄ synthase (LTC₄) andthe leokotriene LTD₄ cause hypotension. The leukotrienes have prominenteffects on the microvasculature. LTC₄ and LTD4 appear to act on theendothelial lining of postcapillary venules to cause exudation ofplasma; they are more potent than histamine in this regard. In higherconcentrations, LTC4 and LTD4 constrict arterioles and reduce exudationof plasma.

[0012] Blood/Platelets

[0013] Prostanoids including prostaglandins and thromboxanes exhibit awide variety of actions in various cells and tissues to maintain localhomeostasis in the body. Eicosanoids modify the function of the formedelements of the blood. PGI2 controls the aggregation of platelets invivo and contributes to the antithrombogenic properties of the intactvascular wall.

[0014] TXA2 is a major product of arachidonate metabolism in plateletsand, as a powerful inducer of platelet aggregation and the plateletrelease reaction, is a physiological mediator of platelet aggregation.Pathways of platelet aggregation that are dependent on the generation ofTXA2 are sensitive to the inhibitory action of aspirin, which inhibitsthe cyclooxygenase (COX) pathway. There has been considerable interestin the elucidation of the role played by prostaglandins and TXA2 inplatelet aggregation and thrombosis and by PGI₂ in the prevention ofthese events. The platelet thromboxane pathway is activated markedly inacute coronary artery syndromes and aspirin is beneficial in thesecondary prevention of coronary and cerebrovascular diseases. PGI thatis generated in the vessel wall may be the physiological antagonist ofthis system; it inhibits platelet aggregation and contributes to thenonthrombogenic properties of the endothelium. According to thisconcept, PGI₂ and TXA2 represent biologically opposite poles of amechanism for regulating platelet-vessel wall interaction and theformation of hemostatic plugs and intraarterial thrombi. There isinterest in drugs which inhibit thromboxane synthase and modulate PGI2production.

[0015] Smooth Muscle

[0016] Prostaglandins contract or relax many smooth muscles beside thoseof the vasculature. The leukotrienes contract most smooth muscles. Ingeneral, PGFs and PGD2 contract and PGEs relax bronchial and trachealmuscle. LTC4 and LTD4 are bronchoconstrictors. They act principally onsmooth muscle in peripheral airways and are 1000 times more potent thanhistamine both in vitro and in vivo. They also stimulate bronchial mucussecretion and cause mucosal edema. A complex mixture of chemicalmessengers is released when sensitized lung tissue is challenged by theappropriate antigen. Various prostaglandins and leukotrienes areprominent components of this mixture. Response to the leukotrienesprobably dominates during allergic constriction of the airway. Evidencefor this conclusion is the ineffectiveness of inhibitors ofcycloxygenase and of histaminergic antagonists in the treatment of humanasthma and the protection afforded by leukotriene antagonists in antigeninduced bronchoconstriction. A particularly important role for thecysteinyl-leukotrienes (LTC4, LTD4, and LTE4) has been suggested inpathogenesis of asthma, which is now recognized as a chronicinflammatory condition. They are potent spasmogens causing a contractionof bronchiolar muscle and an increase in mucus secretion.

[0017] Gastric and Intestinal Secretions

[0018] PGEs and PGI2 inhibit gastric acid secretion stimulated byfeeding, histamine or gastrin. Mucus secretion in the stomach and smallintestine is increased by PGEs. These effects help to maintain theintegrity of the gastric mucosa and are referred to as thecytoprotectant properties of PGEs. Furthermore, PGEs and their analogsinhibit gastric damage caused by a variety of ulcerogenic agents andpromote healing of duodenal and gastric ulcers. Cytoprotection is oftherapeutic importance and PGE₁ analogs are used for the prevention ofgastric ulcers.

[0019] Kidney and Urine Formation

[0020] Prostaglandins modulate renal blood flow and may serve toregulate urine formation by both renovascular and tubular effects.Increased biosynthesis of prostaglandins has been associated withBartter's syndrome, a rare disease, characterized by urinary wasting ofK⁺. Leukotrienes have been involved in the pathophysiology of glomerularimmune injury.

[0021] Reproduction and Parturition

[0022] Much interest is attached to the possible involvement ofprostaglandins in reproductive physiology. Lowered concentrations ofprostaglandins in semen have been implicated in male infertility.Prostaglandins are also thought to contribute to the symptoms of primarydysmenorrhea. Inhibitors of cyclooxygenase are effective in relievingthe symptoms of this condition. Elevated levels of prostaglandins areinvolved in onset of labor. Inhibitors of cyclooxygenase increase thelength of gestation and interrupt premature labor.

[0023] Cancer Metastasis

[0024] Tumors in animals and certain spontaneous human tumors areaccompanied by increased concentrations of local or circulatingprostaglandins. Eicosanoids have been shown to be involved in variousaspects of neoplasia including cell transformation, tumor promotion,tumor cell growth, and metastasis. Some studies have implicated plateletaggregation and the effects of prostaglandins andhydroxyeicosatetraenoic acid (12-HETE) in the hematogenous metastasis oftumors.

[0025] Many of the products of arachidonic acid metabolism are potentmediators of physiological responses and contribute to disorders ofdevelopment, cellular function, tissue repair, and host defenses in anumber of diseases.

[0026] Arachidonic Acid Metabolism And Biosynthesis Of Eicosanoids

[0027] The primary source of eicosanoids in mammalian systems is themetabolic products of arachidonic acid. After stimulation by trauma,infection, or inflammation, translocated phospholipases, especiallyphospholipase A₂, act on membrane phospholipids to liberate arachidonicacid. Once released, arachidonate is metabolized to oxygenated productsby several distinct enzyme pathways, including cyclooxygenases, severallipoxygenases, and cytochrome P450s (CYP). The specific enzyme pathwayinvolved determines, which products are formed.

[0028] Release of Arachidonic Acid from Cell Membranes and itsRegulation

[0029] The eicosanoids are a family of substances produced from thepolyunsaturated fatty acid arachidonic acid, which is present inplasma-membrane phospholipids. The first rate-limiting step in thebiosynthesis of eicosanoids is the release of arachidonic acid from themembrane, a process that is mainly catalyzed by cytosolic phosholipaseA₂ (cPLA₂). The synthesis of eicosanoids begins when a stimulus such asa hormone, a neurotransmitter, a drug or a toxic agent activatescytosolic phospholipase A₂. This arachidonic acid specific phospholipaseplays a major role in the cell signaling events that initiate thearachidonate cascade. One important trigger of arachidonate release andeicosanoid synthesis involves tissue injury and inflammation.

[0030] The activities of many enzymes are regulated by calmodulins (CAL)that serve as calcium sensors in eukaryotic cells. The binding of Ca²⁺to multiple sites in calmodulin induces a major conformational changethat converts it from an inactive to an active form. Activatedcalmodulin then binds to many enzymes and target proteins in the cell,modifying their activities and thereby regulating various metabolicpathways. Calmodulins are involved in a number of processes regulated byCa²⁺ including smooth muscle contraction, neurotransmission, apoptosis,cell cycle progression and gene expression. Calmodulins also participatein the regulation of arachidonate release. They directly stimulatecytosolic phospholipase A₂, whereas calmodulin antagonists inhibitenzyme activity and the release of arachidonic acid.

[0031] Annexins (ANX) are a family of multifunctional calcium andphospholipid-binding proteins, they belong to a family of proteins thatinteract with phospholipids in a Ca²⁺ dependant manner.

[0032] Annexins have been implicated in the pathogenesis of benign andmalignant neoplasms of different origins. Moreover, several annexinshave also been involved in autoimmune diseases such as systemic lupuserythematosus, rheumatoid arthritis and inflammatory bowl disease.Numerous physiological functions have been attributed to annexinsincluding regulation of membrane traffic during exocytosis andendocytosis, mediation of cytoskeletal-membrane interactions, membranereceptor function, regulation of membrane-dependent enzymes, mitogenicsignal transduction, transmembrane ion channel activity, cell-celladhesion, antiinflammatory properties, inhibition of blood coagulationand inhibition of phospholipase A₂. Annexins have been suggested asregulators of prostaglandin metabolism and of the arachidonate cascadeas a result of their inhibitory effect on phospholipase A₂. It is stilla matter of debate as to whether inhibition of phospholipase A2 is theresult of calcium-dependent sequestration of phospholipids (substratedepletion mechanism) or a direct effect of the annexins acting viaprotein-protein interactions. Calpactin I (light chain) is the cellularligand of annexin II and induces its dimerization. Annexin II andcalpactin I (CALPA) constitute a calcium binding complex composed of twolight chains (calpactin I) and two heavy chains (annexin II). CalpactinI may function as regulator of annexin II phosphorylation.

[0033] The activities of phospholipase A₂, annexins and calmodulins arecommon points of regulation in the formation of all eicosanoids.

[0034] Downstream of phospholipase A₂, the varying eicosanoid-pathwayenzymes found in particular cell types determine which eicosanoids aresynthesized in response to particular stimuli.

[0035] Cyclooxygenase Pathway

[0036] This pathway initiated by cyclooxygenase (COX) leads ultimatelyto formation of the cyclic endoperoxides, prostaglandins (PG), andthromboxanes (TX). There are two isoforms of the cyclooxygenase, COX-1and COX-2. The former is constitutively expressed in most cells. Incontrast, COX-2 is not normally present but may be induced by certainfactors such as cytokines and growth factors. The cyclooxygenases havetwo distinct activities: an endoperoxidase synthase activity thatoxygenates and cyclizes the unesterified precursor fatty acid to formthe cyclic endoperoxide PGG and a peroxidase activity that converts PGGto PGH. PGG and PGH are chemically unstable, but they can be transformedenzymatically into a variety of products, including PGI, TXA2, PGE, PGFor PGD. Isomerases lead to the synthesis of PGE₂ and PGD₂, whereas PGI₂is formed from PGH₂ through prostacyclin synthase. TXA2 is formed bythromboxane synthase. Although most tissues are able to synthesize thePGG and PGH intermediates from free arachidonate, the fate of theseprecursors varies in each tissue and depends on the complement ofenzymes that are present and on their relative abundance. For example,lung and spleen are able to synthesize the whole range of products. Incontrast, platelets contain thromboxane synthase as the principal enzymethat metabolizes PGH, while endothelial cells contain primarilyprostacyclin synthase.

[0037] Lipoxygenase Pathways

[0038] Lipoxygenases are a family of cytosolic enzymes that catalyze theoxygenation of fatty acids to corresponding lipid hydroperoxides.Arachidonate is metabolized to HPETE (hydroperoxyeicosatetraenoic acid),which is then converted either enzymatically or non-enzymatically to12-HETE (hydroxyeicosatetraenoic acid). HPETEs may further be convertedto hepoxilins and lipoxins. Lipoxygenases differ in their specificityfor placing the hydroperoxy group, and tissues differ in thelipoxygenases they contain. These enzymes are referred to as 12-, 15-,5- and 8-lipoxygenases according to the oxygenation sites in arachidonicacid as substrate.

[0039] The lipoxygenases catalyze reactions and generate products ofpotential relevance to membrane remodeling, cell differentiation andinflammation. Products of the 15-LO pathway could contribute to thepathophysiology of allergic airway inflammation while products of the12-LO pathway have been implicated in cancer metastasis, psoriasis andinflammation.

[0040] Various biological activities have been reported for the12-lipoxygenase metabolites of arachidonic acid. As other eicosanoids,they are important chemical mediators and modulators of the inflammatoryreaction. 12-HETE is the major arachidonic acid metabolite of12-lipoxygenase and seems to be implicated in a wide-spectrum ofbiological activities such as stimulation of insulin secretion bypancreatic tissue, suppression of renin production, chemoattraction ofleukocytes and initiation of growth-related signaling events, such asactivation of oncogenes, protein kinase C, and mitogen-activated proteinkinases. 12-lipoxygenase activity and 12-HETE production are alsoimportant determining factors in tumor cell metastasis and have beenimplicated in human prostate cancer and breast cancer (Honn et al.,Cancer Metastasis Rev.,13:365-396, 1994, Gao et al., Adv. Exp. Med.Biol., 407:41-53, 1997; Natarajan et al., J. Clin. Endocr. Metab.,82:1790-1789, 1997,). Further, 12-HETE has also been implicated ininflammatory skin diseases such as psoriasis (Hussai et al., Am. J.Physiol., 266:243-253, 1994). As mentioned above, metabolism ofarachidonic acid by 12-lipoxygenase further generates lipoxins andhepoxillins. Lipoxins play the role of both immunologic and hemodynamicregulators and a variety of biological activities have been reported forhepoxillins which are related to the release of intracellular calciumand the opening of potassium channels (Yamamoto et al., Pro. Lipid Res.,36:23-41, 1997).

[0041] The 5-lipoxygenase (5- LO) is perhaps the most important of theseenzymes since it leads to the synthesis of leukotrienes. Activation ofthe 5-LO enzyme involves its docking to a protein termed5-lipoxygenase-activating protein (FLAP). This binding activates theenzyme, results in its association with the cell membrane and increasedsynthesis of 5-HPETE and leukotrienes. Leukotriene A (LTA) synthase isassociated with 5-lipoxygenase and promotes the rearrangement of 5-HPETEto an unstable intermediate LTA₄; which may be transformed to LTB₄ byleukotriene A₄ hydrolase (LTA4H); alternatively, it may be conjugatedwith glutathione by LTC₄ synthase to form LTC₄. LTA4 hydrolase is apivotal element in leukotriene biosynthesis. Omega-oxidation is regardedas the major pathway for the catabolism of LTB₄. This reaction iscatalyzed by LTB₄ omega-hydroxylase (LTB4H3) also called CYP4F2. LTD₄ isproduced by the removal of glutamic acid from LTC₄ and LTE₄ results fromthe subsequent cleavage of glycine; the reincorporation of glutamic acidyields LTF₄.

[0042] Epoxygenase Pathway

[0043] Arachidonate is metabolized to a variety of metabolites byenzymes that contain cytochrome P450. The epoxygenase pathway of thearachidonic acid cascade leads to the formation of epoxyeicosatrienoicacids (EETs) and dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is ahuman cytochrome P450 arachidonic acid epoxygenase expressed inextrahepatic tissues and particularly in the intestine. In addition tothe known effects on intestinal vascular tone, CYP2J2 products may beinvolved in the release of intestinal neuropeptides, control ofintestinal motility and modulation of intestinal fluid/electrolytetransport.

[0044] Eicosanoid Receptors

[0045] The diversity of the effects of eicosanoids is explained by theexistence of a number of distinct receptors that mediate their actions.All prostaglandin receptors identified to date are coupled to effectormechanisms through G proteins. Distinct receptors for leukotrienes alsohave been identified in different tissues, all of these appear toactivate phospholipase C.

[0046] Therapeutic Agents Interacting with Arachidonic Acid Metabolism

[0047] Because of their involvement in so many disease states, there hasbeen a considerable effort to develop effective inhibitors to theformation or action of the eicosanoids. The drugs that influence theeicosanoid pathways are the most commonly used drugs in the world today.Their major uses are to reduce pain, fever and inflammation. Severalclasses of drugs, most notably the nonsteroidal antiinflammatory drugs(NSAIDs) owe their therapeutic effects to blockade of the formation ofeicosanoids. Selective inhibitors of arachidonic acid metabolism alsohave an important therapeutic value. Inhibition of cyclooxygenase (COX),the enzyme responsible for the biosynthesis of the prostaglandins andcertain related autacoids, generally is thought to be a major facet ofthe mechanism of NSAIDs. Aspirin and newer, widely used drugs belong tothe NSAIDs. All NSAIDs are antipyretic, analgesic and antiinflammatorybut there are important differences in their activities and in theirside effects. The reasons for such differences are not fully understood.Side effects of these drugs include gastrointestinal ulceration,disturbances in platelet function, changes in renal function andhypersensitivity reactions. It is now appreciated that there are twoforms of cyclooxygenase (COX), inhibition of COX-2 is thought to mediatethe antipyretic, analgesic and antiinflammatory action of NSAIDs,whereas the simultaneous inhibition of COX-1 may result in unwanted sideeffects. Efforts are under way to identify COX-2 specific agents. But,it is also possible that enhanced generation of lipoxygenase products,due to the diversion of arachidonic acid metabolism from thecyclooxygenase pathway towards the lipoxygenase pathways, contributes tosome of the side effects. Effort is being devoted to a search for drugsthat will produce more selective interventions by acting farther alongthe biosynthetic pathways. Several compounds have been described thatselectively antagonize responses to TXA2 and to PGH₂. Some are receptorantagonists others directly inhibit thromboxane synthase.

[0048] Advances in understanding the pathobiology of the inflammatoryprocess has suggested several novel approaches for development of drugsto block this process. These include phospholipase A₂ inhibitors.Glucocorticoids are thought to have an effect on arachidonic acidmetabolism through the induction of lipocortin that inhibitsphospholipase A₂.

[0049] NSAIDs generally do not inhibit the formation of othereicosanoids such as the lipoxygenase-produced leukotrienes. Substantialevidence indicates that leukotrienes contribute to the inflammatoryresponse through a variety of effects. Leukotrienes have been implicatedas mediators of inflammation and immediate hypersensitivity reactions—inparticular, human bronchial asthma—and thus considerable effort has beendone to develop either inhibitors of the production or blockers of theaction of the actions of these mediators. Various therapeutic approacheshave been used including 5-lipoxygenase inhibitors, which blockleukotriene formation, or cysteinyl leukotriene receptor antagonists,which block receptor function. LTC₄ synthase is another key step inbiosynthesis of leukotrienes and represents another possible site fortherapeutic intervention. Drugs targeting leukotriene biosynthesis arebeing tested and used for their utility in the treatment of variousinflammatory conditions.

[0050] Most of these drugs are efficacious in providing relief but allavailable agents have associated, and sometimes severe, toxicity.Certain individuals display intolerance to aspirin and to other drugsacting on arachidonic acid metabolism; this is manifest by symptoms thatrange from liver toxicity, gastric and intestinal ulceration,disturbance in platelet function, renal injury, nephritis, vasomotorrhinitis with profuse watery secretions, angioneurotic edema,generalized urticaria, and bronchial asthma to laryngeal edema andbronchoconstriction, hypotension, and shock. The underlying mechanismfor these severe side effects is not known. Moreover, while these agentshave been highly useful for treatment of acute, self-limitedinflammatory conditions; their ability to modify disease progression inchronic inflammatory settings remains an area of controversy. Thecomplexity of the highly regulated pathways and enzymes that lead to theformation of the eicosanoids, has limited the precise identification ofthe metabolites and enzymes in the arachidonic acid cascade, which playthe causal role in pathologies or in side effects to some drugs.

[0051] Pharmacogenomics and Arachidonic Acid Metabolism

[0052] The vast majority of common diseases, such as cancer,hypertension, diabetes and some inflammatory diseases are polygenic,meaning that they are caused by multiple genes. In addition, thesediseases are modulated by environmental factors such as pollutants,chemicals and diet. This is why many diseases are called multifactorial;they result from a synergistic combination of factors, both genetic andenvironmental. Therapeutic management and drug development could bemarkedly improved by the identification of specific geneticpolymorphisms that determine and predict patient susceptibility todiseases or patient responses to drugs.

[0053] To assess the origins of individual variations in diseasesusceptibility or drug response, pharmacogenomics uses the genomictechnologies to identify polymorphisms within genes which are part ofbiological pathways involved in disease susceptibility, etiology, anddevelopment, or more specifically in drug response pathways responsiblefor a drug's efficacy, tolerance or toxicity. It can provide tools torefine the design of drug development by decreasing the incidence ofadverse events in drug tolerance studies, by better defining patientsubpopulations of responders and non-responders in efficacy studies and,by combining the results obtained therefrom, to further allow betterenlightened individualized drug usage based on efficacy/toleranceprognosis. Pharmacogenomics can also provide tools to identify newtargets for designing drugs and to optimize the use of already existingdrugs, in order to either increase their response rate and/or excludenon-responders from corresponding treatment, or decrease theirundesirable side effects and/or exclude from corresponding treatmentpatients with marked susceptibility to undesirable side effects.However, for pharmacogenomics to become clinically useful on a largescale, molecular tools and diagnostics tests must become available.

[0054] Inflammatory reactions, which are involved in numerous diseases,are highly relevant to pharmacogenomics both because they are at thecore of many widespread serious diseases, and because targetinginflammation pathways to design new efficient drugs includes numerousrisks of potentiating serious side effects. Arachidonic acid metabolismis particularly relevant since its products, the eicosanoids, arepowerful inflammatory molecules and play a role in a number ofphysiological functions.

[0055] Genetic Analysis of Complex Traits

[0056] Until recently, the identification of genes linked withdetectable traits has relied mainly on a statistical approach calledlinkage analysis. Linkage analysis is based upon establishing acorrelation between the transmission of genetic markers and that of aspecific trait throughout generations within a family. Linkage analysisinvolves the study of families with multiple affected individuals and isuseful in the detection of inherited-traits, which are caused by asingle gene, or possibly a very small number of genes. Linkage analysishas been successfully applied to map simple genetic traits that showclear Mendelian inheritance patterns and which have a high penetrance(the probability that a person with a given genotype will exhibit atrait). About 100 pathological trait-causing genes have been discoveredusing linkage analysis over the last 10 years.

[0057] But, linkage studies have proven difficult when applied tocomplex genetic traits. Most traits of medical relevance do not followsimple Mendelian monogenic inheritance. However, complex diseases oftenaggregate in families, which suggests that there is a genetic componentto be found. Such complex traits are often due to the combined action ofmultiple genes as well as environmental factors. Such complex trait,include susceptibilities to heart disease, hypertension, diabetes,cancer and inflammatory diseases. Drug efficacy, response andtolerance/toxicity can also be considered as multifactoral traitsinvolving a genetic component in the same way as complex diseases.Linkage analysis cannot be applied to the study of such traits for whichno large informative families are available. Moreover, because of theirlow penetrance, such complex traits do not segregate in a clear-cutMendelian manner as they are passed from one generation to the next.Attempts to map such diseases have been plagued by inconclusive results,demonstrating the need for more sophisticated genetic tools.

[0058] Knowledge of genetic variation in the arachidonic acid cascade isimportant for understanding why some people are more susceptible todisease involving arachidonic acid metabolites or respond differently totreatments targeting arachidonic acid metabolism. Ways to identifygenetic polymorphism and to analyze how they impact and predict diseasesusceptibility and response to treatment are needed.

[0059] Although the genes involved in arachidonic acid metabolismrepresent major drug targets and are of high relevance to pharmaceuticalresearch, we still have scant knowledge concerning the extent and natureof sequence variation in these genes and their regulatory elements. Forexample, the cDNA and part of the genomic sequence for human12-lipoxygenase have been cloned and sequenced (Izumi et al., Proc.Natl. Acad. Sci. USA, 87:7477-7481, 1990; Funk et al., Proc. Natl. Acad.Sci. USA, 87:5638-5642, 1990; Yoshimoto et al., Biochem. Biophys. Res.Commun., 172:1230-1235, 1990, Yoshimoto, et al., J. Biol. Chem.,267:24805-24809, 1992). However, the complete genomic sequence of the12-lipoxygenase, including its regulatory elements, have not beendescribed.

[0060] In the cases where polymorphisms have been identified, therelevance of the variation is rarely understood. While polymorphismshold promise for use as genetic markers in determining which genescontribute to multigenic or quantitative traits, suitable markers andsuitable methods for exploiting those markers have not been found andbrought to bare on the genes related to arachidonic acid metabolism.

SUMMARY OF THE INVENTION

[0061] The present invention is based on the discovery of a set of noveleicosanoid-related biallelic markers. See Table 7(A-B). These markersare located in the coding regions as well as non-coding regions adjacentto genes which express proteins associated with arachidonic acidmetabolism. The position of these markers and knowledge of thesurrounding sequence has been used to design polynucleotide compositionswhich are useful in determining the identity of nucleotides at themarker position, as well as more complex association and haplotypingstudies which are useful in determining the genetic basis for diseasestates involving arachidonic acid metabolism. In addition, thecompositions and methods of the invention find use in the identificationof the targets for the development of pharmaceutical agents anddiagnostic methods, as well as the characterization of the differentialefficacious responses to and side effects from pharmaceutical agentsacting on arachidonic acid metabolism.

[0062] The present invention further stems from the isolation andcharacterization of the genomic sequence of the 12-lipoxygenase geneincluding its regulatory regions and of the complete cDNA sequenceencoding the 12-lipoxygenase enzyme. Oligonucleotide probes and primershybridizing specifically with a genomic sequence of 12-lipoxygenase arealso part of the invention. Furthermore, an object of the inventionconsists of recombinant vectors comprising any of the nucleic acidsequences described in the present invention, and in particular ofrecombinant vectors comprising the promoter region of 12-lipoxygenase ora sequence encoding the 12-lipoxygenase enzyme, as well as cell hostscomprising said nucleic acid sequences or recombinant vectors. Theinvention also encompasses methods of screening of molecules which,modulate or inhibit the expression of the 12-lipoxygenase gene. Theinvention is also directed to biallelic markers that are located withinthe 12-lipoxygenase genomic sequence, these biallelic markersrepresenting useful tools in order to identify a statisticallysignificant association between specific alleles of 12-lipoxygenase geneand one or several disorders related to asthma and/or hepatotoxicity.

[0063] A first embodiment of the invention encompasses polynucleotidesconsisting of, consisting essentially of, or comprising a contiguousspan of nucleotides of a sequence selected as an individual or in anycombination from the group consisting of SEQ ID Nos. 1-70, 72-418,425-489, 491-530, 532-539, and 541-652, and the complements thereof;preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184,1186-1193, and 1195-1304, and the complements thereof, or morepreferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143,1145-1184, 1186-1193, and 1195 -1300, and the complements thereof,wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30,35, 40, 50, 75, 100, 200, 500, or 1000 nucleotides in length, to theextent that such a length is consistent with the lengths of theparticular Sequence ID. The present invention also relates topolynucleotides hybridizing under stringent or intermediate conditionsto a sequence selected as an individual or in any combination from thegroup consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539,and 541-652, and the complements thereof, preferably SEQ ID Nos.651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304, and the complements thereof; or more preferably from SEQ IDNos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300, and the complements thereof. In addition, the polynucleotidesof the invention encompass polynucleotides with any further limitationdescribed in this disclosure, or those following, specified alone or inany combination: Said contiguous span may optionally include theeicosanoid-related biallelic marker in said sequence; Optionally eitherthe original or the alternative allele of Table 9 may be specified asbeing present at said eicosanoid-related biallelic marker; Optionallyeither the first or the second allele of Tables 8 or 10 may be specifiedas being present at said eicosanoid-related biallelic marker;Optionally, said polynucleotide may consists of, or consist essentiallyof a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including aneicosanoid-related biallelic marker of said sequence, and optionally theoriginal allele of Table 9 is present at said biallelic marker;Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1nucleotides of the center of said polynucleotide or at the center ofsaid polynucleotide; Optionally, the 3′ end of said contiguous span maybe present at the 3′ end of said polynucleotide; Optionally, biallelicmarker may be present at the 3′ end of said polynucleotide; Optionally,the 3′ end of said polynucleotide may be located within or at least 2,4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotidesupstream of an eicosanoid-related biallelic marker in said sequence, tothe extent that such a distance is consistent with the lengths of theparticular Sequence ID; Optionally, the 3′ end of said polynucleotidemay be located 1 nucleotide upstream of an eicosanoid-related biallelicmarker in said sequence; and Optionally, said polynucleotide may furthercomprise a label.

[0064] A second embodiment of the invention encompasses anypolynucleotide of the invention attached to a solid support. Inaddition, the polynucleotides of the invention which are attached to asolid support encompass polynucleotides with any further limitationdescribed in this disclosure, or those following, specified alone or inany combination: Optionally, said polynucleotides may be specified asattached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20,or 25 distinct polynucleotides of the inventions to a single solidsupport; Optionally, polynucleotides other than those of the inventionmay attached to the same solid support as polynucleotides of theinvention; Optionally, when multiple polynucleotides are attached to asolid support they may be attached at random locations, or in an orderedarray; Optionally, said ordered array may be addressable.

[0065] A third embodiment of the invention encompasses the use of anypolynucleotide for, or any polynucleotide for use in, determining theidentity of one or more nucleotides at an eicosanoid-related biallelicmarker. In addition, the polynucleotides of the invention for use indetermining the identity of one or more nucleotides at aneicosanoid-related biallelic marker encompass polynucleotides with anyfurther limitation described in this disclosure, or those following,specified alone or in any combination. Optionally, saideicosanoid-related biallelic marker may be in a sequence selectedindividually or in any combination from the group consisting of SEQ IDNos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and thecomplements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072,1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complementsthereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072,1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complementsthereof; Optionally, said polynucleotide may comprise a sequencedisclosed in the present specification; Optionally, said polynucleotidemay consist of, or consist essentially of any polynucleotide describedin the present specification; Optionally, said determining may beperformed in a hybridization assay, sequencing assay, microsequencingassay, or an enzyme-based mismatch detection assay; Optionally, saidpolynucleotide may be attached to a solid support, array, or addressablearray; Optionally, said polynucleotide may be labeled.

[0066] A fourth embodiment of the invention encompasses the use of anypolynucleotide for, or any polynucleotide for use in, amplifying asegment of nucleotides comprising an eicosanoid-related biallelicmarker. In addition, the polynucleotides of the invention for use inamplifiing a segment of nucleotides comprising an eicosanoid-relatedbiallelic marker encompass polynucleotides with any further limitationdescribed in this disclosure, or those following, specified alone or inany combination: Optionally, said eicosanoid-related biallelic markermay be in a sequence selected individually or in any combination fromthe group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530,532-539, and 541-652, and the complements thereof; preferably SEQ IDNos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304, and the complements thereof; or more preferably from SEQ IDNos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300, and the complements thereof; Optionally, said polynucleotidemay comprise a sequence disclosed in the present specification;Optionally, said polynucleotide may consist of, or consist essentiallyof any polynucleotide described in the present specification;Optionally, said amplifying may be performed by a PCR or LCR.Optionally, said polynucleotide may be attached to a solid support,array, or addressable array. Optionally, said polynucleotide may belabeled.

[0067] A fifth embodiment of the invention encompasses methods ofgenotyping a biological sample comprising determining the identity of anucleotide at an eicosanoid-related biallelic marker. In addition, thegenotyping methods of the invention encompass methods with any furtherlimitation described in this disclosure, or those following, specifiedalone or in any combination: Optionally, said eicosanoid-relatedbiallelic marker may be in a sequence selected individually or in anycombination from the group consisting of SEQ ID Nos. 1-70, 72-418,425-489, 491-530, 532-539, and 541-652, and the complements thereof;preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184,1186-1193, and 1195-1304, and the complements thereof; or morepreferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143,1145-1184, 1186-1193, and 1195-1300, and the complements thereof,Optionally, said method further comprises determining the identity of asecond nucleotide at said biallelic marker, wherein said firstnucleotide and second nucleotide are not base paired (by Watson & Crickbase pairing) to one another; Optionally, said biological sample isderived from a single individual or subject; Optionally, said method isperformed in vitro; Optionally, said biallelic marker is determined forboth copies of said biallelic marker present in said individual'sgenome; Optionally, said biological sample is derived from multiplesubjects or individuals; Optionally, said method further comprisesamplifying a portion of said sequence comprising the biallelic markerprior to said determining step; Optionally, wherein said amplifying isperformed by PCR, LCR, or replication of a recombinant vector comprisingan origin of replication and said portion in a host cell; Optionally,wherein said determining is performed by a hybridization assay,sequencing assay, microsequencing assay, or an enzyme-based mismatchdetection assay.

[0068] A sixth embodiment of the invention comprises methods ofestimating the frequency of an allele in a population comprisinggenotyping individuals from said population for an eicosanoid-relatedbiallelic marker and determining the proportional representation of saidbiallelic marker in said population. In addition, the methods ofestimating the frequency of an allele in a population of the inventionencompass methods with any further limitation described in thisdisclosure, or those following, specified alone or in any combination:Optionally, said eicosanoid-related biallelic marker may be in asequence selected individually or in any combination from the groupconsisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and541-652, and the complements thereof; preferably SEQ ID Nos. 651-652,655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, andthe complements thereof; or more preferably from SEQ ID Nos. 651-652,680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, andthe complements thereof; Optionally, determining the frequency of abiallelic marker allele in a population may be accomplished bydetermining the identity of the nucleotides for both copies of saidbiallelic marker present in the genome of each individual in saidpopulation and calculating the proportional representation of saidnucleotide at said eicosanoid-related biallelic marker for thepopulation; Optionally, determining the frequency of a biallelic markerallele in a population may be accomplished by performing a genotypingmethod on a pooled biological sample derived from a representativenumber of individuals, or each individual, in said population, andcalculating the proportional amount of said nucleotide compared with thetotal.

[0069] A seventh embodiment of the invention comprises methods ofdetecting an association between an allele and a phenotype, comprisingthe steps of a) determining the frequency of at least oneeicosanoid-related biallelic marker allele in a case population, b)determining the frequency of said eicosanoid-related biallelic markerallele in a control population and; c) determining whether astatistically significant association exists between said genotype andsaid phenotype. In addition, the methods of detecting an associationbetween an allele and a phenotype of the invention encompass methodswith any further limitation described in this disclosure, or thosefollowing, specified alone or in any combination: Optionally, saideicosanoid-related biallelic marker may be in a sequence selectedindividually or in any combination from the group consisting of SEQ IDNos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and thecomplements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072,1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complementsthereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072,1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complementsthereof; Optionally, said control population may be a trait negativepopulation, or a random population; Optionally, each of steps a) and b)is performed on a single pooled biological sample derived from each ofsaid populations; Optionally, each of said steps a) and b) is performedon a single pooled biological sample derived from each of saidpopulations; Optionally, each of said steps a) and b) is performedseparately on biological samples derived from each individual in saidpopulations; Optionally, said phenotype is a disease involvingarachidonic acid metabolism, a response to an agent acting onarachidonic acid metabolism, or a side effects to an agent acting onarachidonic acid metabolism; Optionally, the identity of the nucleotidesat the biallelic markers in everyone of the following sequences: SEQ IDNos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferablySEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193,and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724,726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determinedin steps a) and b).

[0070] An eighth embodiment of the present invention encompasses methodsof estimating the frequency of a haplotype for a set of biallelicmarkers in a population, comprising the steps of: a) genotyping eachindividual in said population for at least one eicosanoid-relatedbiallelic marker, b) genotyping each individual in said population for asecond biallelic marker by determining the identity of the nucleotidesat said second biallelic marker for both copies of said second biallelicmarker present in the genome; and c) applying a haplotype determinationmethod to the identities of the nucleotides determined in steps a) andb) to obtain an estimate of said frequency. In addition, the methods ofestimating the frequency of a haplotype of the invention encompassmethods with any further limitation described in this disclosure, orthose following, specified alone or in any combination: Optionally saidhaplotype determination method is selected from the group consisting ofasymmetric PCR amplification, double PCR amplification of specificalleles, the Clark method, or an expectation maximization algorithm;Optionally, said second biallelic marker is an eicosanoid-relatedbiallelic marker in a sequence selected from the group consisting of thebiallelic markers of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530,532-539, and 541-652, and the complements thereof; preferably SEQ IDNos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304, and the complements thereof; or more preferably from SEQ IDNos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300, and the complements thereof; Optionally, the identity of thenucleotides at the biallelic markers in everyone of the sequences: SEQID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferablySEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193,and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724,726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determinedin steps a) and b).

[0071] A ninth embodiment of the present invention encompasses methodsof detecting an 10 association between a haplotype and a phenotype,comprising the steps of: a) estimating the frequency of at least onehaplotype in a case population according to a method of estimating thefrequency of a haplotype of the invention; b) estimating the frequencyof said haplotype in a control population according to the method ofestimating the frequency of a haplotype of the invention; and c)determining whether a statistically significant association existsbetween said haplotype and said phenotype. In addition, the methods ofdetecting an association between a haplotype and a phenotype of theinvention encompass methods with any further limitation described inthis disclosure, or those following, specified alone or in anycombination: Optionally, said eicosanoid-related biallelic marker may bein a sequence selected individually or in any combination from the groupconsisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and541-652, and the complements thereof; preferably SEQ ID Nos. 651-652,655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, andthe complements thereof; or more preferably from SEQ ID Nos. 651-652,680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, andthe complements thereof; Optionally, said control population may be atrait negative population, or a random population; Optionally, saidphenotype is a disease involving arachidonic acid metabolism, a responseto an agent acting on arachidonic acid metabolism, or a side effects toan agent acting on arachidonic acid metabolism; Optionally, the identityof the nucleotides at the biallelic markers in everyone of the followingsequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143,1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos.651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300 is included in the estimating steps a) and b).

[0072] A tenth embodiment of the present invention is a method ofadministering a drug or a treatment comprising the steps of: a)obtaining a nucleic acid sample from an individual; b) determining theidentity of the polymorphic base of at least one eicosanoid-relatedbiallelic marker or 12-LO-related biallelic marker according to themethods taught herein which is associated with a positive response tosaid drug or treatment, or at least one eicosanoid-related marker or12-LO-related biallelic marker or which is associated with a negativeresponse to said drug or treatment; and c) administering said drug ortreatment to said individual if said nucleic acid sample contains atleast one biallelic marker associated with a positive response to saiddrug or treatment, or if said nucleic acid sample lacks at least onebiallelic marker associated with a negative response to said drug ortreatment. In addition, the methods of the present invention foradministering a drug or a treatment encompass methods with any furtherlimitation described in this disclosure, or those following, specifiedalone or in any combination: optionally, said eicosanoid-relatedbiallelic marker or 12-LO-related biallelic marker may be in a sequenceselected individually or in any combination from the group consisting ofSEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, andthe complements thereof; preferably SEQ ID Nos. 651-652, 655-724,726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and thecomplements thereof; or more preferably from SEQ ID Nos. 651-652,680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, andthe complements thereof; or optionally, the administering step comprisesadministering the drug or the treatment to the individual if the nucleicacid sample contains said biallelic marker associated with a positiveresponse to the treatment or the drug and the nucleic acid sample lackssaid biallelic marker associated with a negative response to thetreatment or the drug.

[0073] An eleventh embodiment of the present invention is a method ofselecting an individual for inclusion in a clinical trial of a treatmentor drug comprising the steps of: a) obtaining a nucleic acid sample froman individual; b) determining the identity of the polymorphic base of atleast one eicosanoid-related biallelic marker or 12-LO-related biallelicmarker which is associated with a positive response to the treatment orthe drug, or at least one eicosanoid-related biallelic marker or12-LO-related biallelic marker which is associated with a negativeresponse to the treatment or the drug in the nucleic acid sample, and c)including the individual in the clinical trial if the nucleic acidsample contains said eicosanoid-related biallelic marker or12-LO-related biallelic marker associated with a positive response tothe treatment or the drug or if the nucleic acid sample lacks saidbiallelic marker associated with a negative response to the treatment orthe drug. In addition, the methods of the present invention forselecting an individual for inclusion in a clinical trial of a treatmentor drug encompass methods with any further limitation described in thisdisclosure, or those following, specified alone or in any combination:Optionally, said eicosanoid-related biallelic marker or 12-LO-relatedbiallelic marker may be in a sequence selected individually or in anycombination from the group consisting of SEQ ID Nos. 1-70, 72-418,425-489, 491-530, 532-539, and 541-652, and the complements thereof;preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184,1186-1193, and 1195-1304, and the complements thereof, or morepreferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143,1145-1184, 1186-1193, and 1195-1300, and the complements thereof.

[0074] Additional embodiments are set forth in the Detailed Descriptionof the Invention and in the Examples.

BRIEF DESCRIPTION OF THE TABLES

[0075] Table 1 contains the first five markers listed in the sequencelisting and their corresponding SEQ ID numbers.

[0076] Tables 2A-C are a list of 12-LO-related biallelic markers.

[0077] Table 3 is a listing of currently available forensic testingsystems and their characteristics as compared to the method of theinvention.

[0078] Table 4 sets forth the number of biallelic markers (VNTRs) neededto obtain, in mean, a ratio of at least 10⁶ or 10⁸.

[0079] Table 5 provides an indication of the descriminatory potential ofthe systems of the invention.

[0080] Table 6 is a listing of probabilities for several different typesof relationships and likelihood ratios.

[0081] Table 7A is a chart containing a list of all of theeicosanoid-related biallelic markers for each gene with an indication ofthe gene for which the marker is in closest physical proximity, anindication of whether the markers have been validated by microsequencing(with a Y indicating that the markers have been validated bymicrosequencing and an N indicating that it has not), and an indicationof the identity and frequency of the least common allele determined bygenotyping (with a blank left to indicate that the frequency has not yetbeen reported for some markers). The frequencies were determined fromDNA samples collected from a random US Caucasian population. When themarker was determined to be homozygous at the particular location forthe random US Caucasian population, the homozygous bases were recordedin the “Genotyping Least Common Allele Frequency” column of Table 7A.For example, Seq. ID No. 16 was determined to be homozygous G/G at thebiallelic marker position 478 in the US control population, thereforeG/G was recorded in the “Genotyping Least Common Allele Frequency”column.

[0082] Table 7B contains all of the eicosanoid-related biallelic markersprovided in Table 7A; however, they are provided in shorter, easier tosearch sequences of 47 nucleotides. Accordingly, Table 7A begins withSEQ ID No. 1 and ends with SEQ ID No. 654, while Table 7B begins withSEQ ID No. 655 and ends with SEQ ID No. 1604 (SEQ ID Nos. 651-654correspond to the genomic and protein sequences of the invention and arenot repeated in Table 7B). Table 1 contains the first five markerslisted in the sequence listing and their corresponding SEQ ID numbers inTables 7A and 7B to illustrate the relationship between Tables 7A and7B: TABLE 1 BIALLELIC SEQ ID BIALLELIC SEQ ID NO. MARKER NO. IN MARKERBIALLELIC IN TABLE POSITION IN TABLE POSITION IN MARKER ID 7A SEQ ID NO.7B SEQ ID NO. 10-253-118 1 478 655 24 10-253-298 2 478 656 24 10-253-3153 478 657 24 10-499-155 4 478 658 24 10-520-256 5 478 659 24

[0083] Table 7B is the same as Table 7A in that it is a list of all ofthe eicosanoid-related biallelic markers for each gene with anindication of the gene for which the marker is in closest physicalproximity, an indication of whether the markers have been validated bymicrosequencing (with a Y indicating that the markers have beenvalidated by microsequencing and an N indicating that it has not), andan indication of the identity and frequency of the least common alleledetermined by genotyping (with a blank left to indicate that thefrequency has not yet been reported for some markers). However, the“Biallelic Marker Position in SEQ ID No.” for all of theeicosanoid-related biallelic markers provided in Table 7B is position 24(representing the midpoint of the 47 mers that make up Table 7B). Thefrequencies were determined from DNA samples collected from a random USCaucasian population. When the marker was determined to be homozygous atthe particular location for the random US Caucasian population, thehomozygous bases were recorded in the “Genotyping Least Common AlleleFrequency” column of Table 7B. For example, Seq. ID No. 670 wasdetermined to be homozygous G/G at the biallelic marker position 24 inthe US control population, therefore G/G was recorded in the “GenotypingLeast Common Allele Frequency” column.

[0084] Tables 8, 9, and 10 are charts containing lists of theeicosanoid-related biallelic markers. Each marker is described byindicating its SEQ ID, the biallelic marker ID, and the two most commonalleles. Table 8 is a chart containing a list of biallelic markerssurrounded by preferred sequences. In the column labeled, “POSITIONRANGE OF PREFERRED SEQUENCE” of Table 8 regions of particularlypreferred sequences are listed for each SEQ ID, which contain aneicosanoid-related biallelic marker, as well as particularly preferredregions of sequences that do not contain an eicosanoid-related biallelicmarker but, which are in sufficiently close proximity to aneicosanoid-related biallelic marker to be useful as amplification orsequencing primers.

[0085] Table 11 is a chart listing particular sequences that are usefulfor designing some of the primers and probes of the invention. Eachsequence is described by indicating its Sequence ID and the positions ofthe first and last nucleotides (position range) of the particularsequence in the Sequence ID.

[0086] Table 12 is a chart listing microsequencing primers which havebeen used to genotype eicosanoid-related biallelic markers (indicated byan *) and other preferred microsequencing primers for use in genotypingeicosanoid-related biallelic markers. Each of the primers which fallswithin the strand of nucleotides included in the Sequence Listing aredescribed by indicating their Sequence ID number and the positions ofthe first and last nucleotides (position range) of the primers in theSequence ID. Since the sequences in the Sequence Listing are singlestranded and half the possible microsequencing primers are composed ofnucleotide sequences from the complementary strand, the primers that arecomposed of nucleotides in the complementary strand are described byindicating their SEQ ID numbers and the positions of the first and lastnucleotides to which they are complementary (complementary positionrange) in the Sequence ID.

[0087] Table 13 is a chart listing amplification primers which have beenused to amplify polynucleotides containing one or moreeicosanoid-related biallelic markers. Each of the primers which fallswithin the strand of nucleotides included in the Sequence Listing aredescribed by indicating their Sequence ID number and the positions ofthe first and last nucleotides (position range) of the primers in theSequence ID. Since the sequences in the Sequence Listing are singlestranded and half the possible amplification primers are composed ofnucleotide sequences from the complementary strand, the primers that arecomposed of nucleotides in the complementary strand are defined by theSEQ ID numbers and the positions of the first and last nucleotides towhich they are complementary (complementary position range) in theSequence ID.

[0088] Table 14 is a chart listing preferred probes useful in genotypingeicosanoid-related biallelic markers by hybridization assays. The probesare 25-mers with an eicosanoid-related biallelic marker in the centerposition, and described by indicating their Sequence ID number and thepositions of the first and last nucleotides (position range) of theprobes in the Sequence ID. The probes complementary to the sequences ineach position range in each Sequence ID are also understood to be a partof this preferred list even though they are not specified separately.

[0089] Table 15 is a table showing the results of the association studybetween biallelic marker haplotypes from the FLAP gene and asthma.

[0090] Table 16 is a table showing the results of the permutation testconfirming the statistical significance of the association betweenasthma and biallelic marker haplotypes from the FLAP gene.

[0091] Table 17 is a table showing the results of the association studybetween 12 biallelic marker haplotypes from the 12-LO gene and asthma.

[0092] Table 18A is a table showing the results of allele frequencyanalysis between seventeen 12-LO biallelic markers and asthma.

[0093] Table 18B is a table showing the results of the association studybetween seventeen 12-LO biallelic marker haplotypes from the 12-LO geneand asthma.

[0094] Table 19 is a table showing the results of the association studybetween 12 biallelic marker haplotypes from the 12-LO gene andhepatotoxicity upon treatment with zileuton.

[0095] Table 20A is a table showing the results of the allele frequencyanalysis between seventeen 12-LO biallelic markers and hepatotoxicityupon treatment with zileuton.

[0096] Table 20B is a table showing the results of the association studybetween seventeen 12-LO biallelic marker haplotypes from the 12-LO geneand hepatotoxicity upon treatment with zileuton.

[0097] Table 21 is a table showing a summary of the association studyresults, permutation tests confirming the statistical significance ofthe association between asthma and biallelic marker haplotypes from the12-LO gene, and permutation tests confirming the statisticalsignificance of the association between secondary effects upon treatmentwith zileuton and biallelic marker haplotypes from the 12-LO gene.

[0098] Table 22 is a table showing a summary of the association studyresults, permutation tests confirming the statistical significance ofthe association between asthma and additional biallelic markerhaplotypes from the 12-LO gene, and permutation tests confirming thestatistical significance of the association between secondary effectsupon treatment with zileuton and biallelic marker haplotypes from the12-LO gene.

[0099] Table 23 is a chart containing a list of preferred 12-LO-relatedbiallelic markers with an indication of the frequency of the leastcommon allele determined by genotyping. Frequencies were determined in arandom US Caucasian population, in an asthmatic population showing noside effects upon treatment with Zyflo™ (ALT−) and in an asthmaticpopulation showing elevated alanine aminotransferase levels upontreatment with Zyflo™ (ALT+).

BRIEF DESCRIPTION OF THE DRAWINGS

[0100]FIG. 1 is a diagram showing the genomic structure of the FLAP geneand the positions of biallelic markers in close proximity of this gene.

[0101]FIG. 2 is a graph showing the results of the single pointassociation study between biallelic markers from the FLAP gene andasthma.

[0102]FIG. 3 is a diagram showing the genomic structure of the12-lipoxygenase gene and the positions of biallelic markers in closeproximity of this gene.

DETAILED DESCRIPTION OF THE INVENTION

[0103] Advantages of the Biallelic Markers of the Present Invention

[0104] The eicosanoid-related biallelic markers of the present inventionoffer a number of important advantages over other genetic markers suchas RFLP (Restriction fragment length polymorphism) and VNTR (VariableNumber of Tandem Repeats) markers.

[0105] The first generation of markers, were RFLPs, which are variationsthat modify the length of a restriction fragment. But methods used toidentify and to type RFLPs are relatively wasteful of materials, effort,and time. The second generation of genetic markers were VNTRs, which canbe categorized as either minisatellites or microsatellites.Minisatellites are tandemly repeated DNA sequences present in units of5-50 repeats which are distributed along regions of the humanchromosomes ranging from 0.1 to 20 kilobases in length. Since theypresent many possible alleles, their informative content is very high.Minisatellites are scored by performing Southern blots to identify thenumber of tandem repeats present in a nucleic acid sample from theindividual being tested. However, there are only 10⁴ potential VNTRsthat can be typed by Southern blotting. Moreover, both RFLP and VNTRmarkers are costly and time-consuming to develop and assay in largenumbers.

[0106] Single nucleotide polymorphism or biallelic markers can be usedin the same manner as RFLPs and VNTRs but offer several advantages.Single nucleotide polymorphisms are densely spaced in the human genomeand represent the most frequent type of variation. An estimated numberof more than 10⁷ sites are scattered along the 3×10⁹ base pairs of thehuman genome. Therefore, single nucleotide polymorphism occur at agreater frequency and with greater uniformity than RFLP or VNTR markerswhich means that there is a greater probability that such a marker willbe found in close proximity to a genetic locus of interest. Singlenucleotide polymorphisms are less variable than VNTR markers but aremutationally more stable.

[0107] Also, the different forms of a characterized single nucleotidepolymorphism, such as the biallelic markers of the present invention,are often easier to distinguish and can therefore be typed easily on aroutine basis. Biallelic markers have single nucleotide based allelesand they have only two common alleles, which allows highly paralleldetection and automated scoring. The biallelic markers of the presentinvention offer the possibility of rapid, high-throughput genotyping ofa large number of individuals.

[0108] Biallelic markers are densely spaced in the genome, sufficientlyinformative and can be assayed in large numbers. The combined effects ofthese advantages make biallelic markers extremely valuable in geneticstudies. Biallelic markers can be used in linkage studies in families,in allele sharing methods, in linkage disequilibrium studies inpopulations, in association studies of case-control populations. Animportant aspect of the present invention is that biallelic markersallow association studies to be performed to identify genes involved incomplex traits. Association studies examine the frequency of markeralleles in unrelated case- and control-populations and are generallyemployed in the detection of polygenic or sporadic traits. Associationstudies may be conducted within the general population and are notlimited to studies performed on related individuals in affected families(linkage studies). Biallelic markers in different genes can be screenedin parallel for direct association with disease or response to atreatment. This multiple gene approach is a powerful tool for a varietyof human genetic studies as it provides the necessary statistical powerto examine the synergistic effect of multiple genetic factors on aparticular phenotype, drug response, sporadic trait, or disease statewith a complex genetic etiology.

[0109] Candidate Genes of the Present Invention

[0110] Different approaches can be employed to perform associationstudies: genome-wide association studies, candidate region associationstudies and candidate gene association studies. Genome-wide associationstudies rely on the screening of genetic markers evenly spaced andcovering the entire genome. Candidate region association studies rely onthe screening of genetic markers evenly spaced covering a regionidentified as linked to the trait of interest. The candidate geneapproach is based on the study of genetic markers specifically derivedfrom genes potentially involved in a biological pathway related to thetrait of interest. In the present invention, genes involved inarachidonic acid metabolism have been chosen as candidate genes. Thismetabolic pathway leads to the biosynthesis of eicosanoids, which arechemical mediators that play an important role in a number ofinflammatory diseases, moreover, these pathways are important drugtargets and genetic polymorphisms in these genes are highly relevant inthe response to a number of drugs. The candidate gene analysis clearlyprovides a short-cut approach to the identification of genes and genepolymorphisms related to a particular trait when some informationconcerning the biology of the trait is available as is the case forarachidonic acid metabolism. However, it should be noted that all of thebiallelic markers disclosed in the instant application can be employedas part of genome-wide association studies or as part of candidateregion association studies and such uses are specifically contemplatedin the present invention and claims. All of the markers are known to bein close proximity to the genes with which they are listed in Table 7.For a portion of the markers, the precise position of the marker withrespect to the various coding and non-coding elements of the genes hasalso been determined.

[0111] Definitions

[0112] As used interchangeably herein, the terms “oligonucleotides”,“nucleic acids” and “polynucleotides” include RNA, DNA, or RNA/DNAhybrid sequences of more than one nucleotide in either single chain orduplex form. The term “nucleotide” as used herein as an adjective todescribe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences ofany length in single-stranded or duplex form. The term “nucleotide” isalso used herein as a noun to refer to individual nucleotides orvarieties of nucleotides, meaning a molecule, or individual unit in alarger nucleic acid molecule, comprising a purine or pyrimidine, aribose or deoxyribose sugar moiety, and a phosphate group, orphosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. Although the term “nucleotide” isalso used herein to encompass “modified nucleotides” which comprise atleast one modifications (a) an alternative linking group, (b) ananalogous form of purine, (c) an analogous form of pyrimidine, or (d) ananalogous sugar, for examples of analogous linking groups, purine,pyrimidines, and sugars see for example PCT publication No. WO 95/04064.However, the polynucleotides of the invention are preferably comprisedof greater than 50% conventional deoxyribose nucleotides, and mostpreferably greater than 90% conventional deoxyribose nucleotides. Thepolynucleotide sequences of the invention may be prepared by any knownmethod, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

[0113] Throughout the present specification, the expression “nucleotidesequence” may be employed to designate indifferently a polynucleotide ora nucleic acid. More precisely, the expression “nucleotide sequence”encompasses the nucleic material itself and is thus not restricted tothe sequence information (i.e. the succession of letters chosen amongthe four base letters) that biochemically characterizes a specific DNAor RNA molecule.

[0114] The term “polypeptide” refers to a polymer of amino withoutregard to the length of the polymer; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude prost-expression modifications ofpolypeptides, for example, polypeptides which include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

[0115] The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

[0116] As used herein, the term “isolated” requires that the material beremoved from its original environment (e.g., the natural environment ifit is naturally occurring). For example, a naturally-occurringpolynucleotide present in a living animal is not isolated, but the samepolynucleotide, separated from some or all of the coexisting materialsin the natural system, is isolated. Specifically excluded from thedefinition of “isolated” are: naturally occurring chromosomes (e.g.,chromosome spreads) artificial chromosome libraries, genomic libraries,and cDNA libraries that exist either as an in vitro nucleic acidpreparation or as a transfected/transformed host cell preparation,wherein the host cells are either an in vitro heterogeneous preparationor plated as a heterogeneous population of single colonies. Alsospecifically excluded are the above libraries wherein the 5′ EST makesup less than 5% of the number of nucleic acid inserts in the vectormolecules. Further specifically excluded are whole cell genomic DNA orwhole cell RNA preparations (including said whole cell preparationswhich are mechanically sheared or enzymaticly digested). Furtherspecifically excluded are the above whole cell preparations as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis (including blot transfers of the same) wherein thepolynucleotide of the invention have not been further separated from theheterologous polynucleotides in the electrophoresis medium (e.g.,further separating by excising a single band from a heterogeneous bandpopulation in an agarose gel or nylon blot).

[0117] As used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition. Individual 5′EST clones isolated from a cDNA library have been conventionallypurified to electrophoretic homogeneity. The sequences obtained fromthese clones could not be obtained directly either from the library orfrom total human DNA. The cDNA clones are not naturally occurring assuch, but rather are obtained via manipulation of a partially purifiednaturally occurring substance (messenger RNA). The conversion of mRNAinto a cDNA library involves the creation of a synthetic substance(cDNA) and pure individual cDNA clones can be isolated from thesynthetic library by clonal selection. Thus, creating a cDNA libraryfrom messenger RNA and subsequently isolating individual clones fromthat library results in an approximately 10⁴-10⁶ fold purification ofthe native message. Purification of starting material or naturalmaterial to at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated. Alternatively, purification may be expressed as“at least” a percent purity relative to heterologous polynucleotides(DNA, RNA or both). As a preferred embodiment, the polynucleotides ofthe present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologouspolynucleotides. As a further preferred embodiment the polynucleotideshave an “at least” purity ranging from any number, to the thousandthposition, between 90% and 100% (e.g., 5′ EST at least 99.995% pure)relative to heterologous polynucleotides. Additionally, purity of thepolynucleotides may be expressed as a percentage (as described above)relative to all materials and compounds other than the carrier solution.Each number, to the thousandth position, may be claimed as individualspecies of purity. The term “primer” denotes a specific oligonucleotidesequence which is complementary to a target nucleotide sequence and usedto hybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by DNApolymerase, RNA polymerase or reverse transcriptase.

[0118] The term “probe” denotes a defined nucleic acid segment (ornucleotide analog segment, e.g., polynucleotide as defined herein) whichcan be used to identify a specific polynucleotide sequence present insamples, said nucleic acid segment comprising a nucleotide sequencecomplementary of the specific polynucleotide sequence to be identified.

[0119] The term “disease involving arachidonic acid metabolism” refersto a condition linked to disturbances in expression, production orcellular response to eicosanoids such as prostaglandins, thromboxanes,prostacyclins, leukotrienes or hydroperoxyeicosaetrenoic acids. Adisease involving arachidonic acid metabolism further refers to acondition involving one or several enzymes of the distinct enzymesystems contributing to arachidonate metabolism including particularlythe cyclooxygenase pathway and the lipoxygenase pathway and thearachadonic acid metabolites of such systems including 12-HETE,12-HPETE, lipoxins and hepoxolins. “Diseases involving arachidonic acidmetabolism” also include chronic inflammatory diseases, acute allergicinflammation and inflammatory conditions such as pain, fever,hypersensitivity, asthma, psoriasis and arthritis. “Diseases involvingarachidonic acid metabolism” also include disorders in plateletfunction, blood pressure, thrombosis, renal function, host defensemechanism, hemostasis, smooth muscle tone, male infertility, primarydysmenorrhea, disorders in parturition, and disorders in tissue injuryrepair, as well as disorders in cellular function and development.“Diseases involving arachidonic acid metabolism” also include diseasessuch as gastrointestinal ulceration, coronary and cerebrovascularsyndromes, glomerular immune injury and cancer.

[0120] The term “agent acting on arachidonic acid metabolism” refers toa drug or a compound modulating the activity or concentration of anenzyme or regulatory molecule involved in arachidonic acid metabolism,including but not limited to cyclooxygenase, prostacyclin synthase,thromboxane synthase, lipoxygenases, 5-lipoxygenase and 5-lipoxygenaseactivating protein. “Agent acting on arachidonic acid metabolism”further refers to non-steroidal antiinflammatory drugs (NSAIDs),eicosanoid receptor antagonists, eicosanoid analogs, COX-1 inhibitors,COX-2 inhibitors, thromboxane synthase inhibitors, 5-lipoxygenaseinhibitors and 5-lipoxygenase activating protein inhibitors. “Agentacting on arachidonic acid metabolism” also refers to compoundsmodulating the formation and action of eicosanoids such asprostaglandins, prostacyclins, thromboxanes, leukotrienes orhydroperoxyeicosaetrenoic acids.

[0121] The terms “response to an agent acting on arachidonic acidmetabolism” refer to drug efficacy, including but not limited to abilityto metabolize a compound, to the ability to convert a pro-drug to anactive drug, and to the pharmacokinetics (absorption, distribution,elimination) and the pharmacodynamics (receptor-related) of a drug in anindividual.

[0122] The terms “side effects to an agent acting on arachidonic acidmetabolism” refer to adverse effects of therapy resulting fromextensions of the principal pharmacological action of the drug or toidiosyncratic adverse reactions resulting from an interaction of thedrug with unique host factors. “Side effects to an agent acting onarachidonic acid metabolism” include, but are not limited to, adversereactions such as dermatologic, hematologic or hepatologic toxicitiesand further includes gastric and intestinal ulceration, disturbance inplatelet function, renal injury, nephritis, vasomotor rhinitis withprofuse watery secretions, angioneurotic edema, generalized urticaria,and bronchial asthma to laryngeal edema and bronchoconstriction,hypotension, and shock.

[0123] The terms “trait” and “phenotype” are used interchangeably hereinand refer to any visible, detectable or otherwise measurable property ofan organism such as symptoms of, or susceptibility to a disease forexample. Typically the terms “trait” or “phenotype” are used herein torefer to symptoms of, or susceptibility to a disease involvingarachidonic acid metabolism; or to refer to an individual's response toan agent acting on arachidonic acid metabolism; or to refer to symptomsof, or susceptibility to side effects to an agent acting on arachidonicacid metabolism.

[0124] The terms “agent acting on 5-lipoxygenase” refers to a drug or acompound modulating the activity or concentration of the 5-lipoxygenaseenzyme such as 5-lipoxygenase inhibitors. “Agent acting on5-lipoxygenase” also refers to compounds modulating the formation andaction of leukotrienes.

[0125] The terms “side effects to an agent acting on 5-lipoxygenase”include, but are not limited to, adverse reactions such as dermatologic,hematologic or hepatologic toxicities.

[0126] The term “allele” is used herein to refer to variants of anucleotide sequence. A biallelic polymorphism has two forms. Typicallythe first identified allele is designated as the original allele whereasother alleles are designated as alternative alleles. Diploid organismsmay be homozygous or heterozygous for an allelic form.

[0127] The term “heterozygosity rate” is used herein to refer to theincidence of individuals in a population, which are heterozygous at aparticular allele. In a biallelic system the heterozygosity rate is onaverage equal to 2P_(a)(1−P_(a)), where P_(a) is the frequency of theleast common allele. In order to be useful in genetic studies a geneticmarker should have an adequate level of heterozygosity to allow areasonable probability that a randomly selected person will beheterozygous.

[0128] The term “genotype” as used herein refers the identity of thealleles present in an individual or a sample. In the context of thepresent invention a genotype preferably refers to the description of thebiallelic marker alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a biallelic marker consistsof determining the specific allele or the specific nucleotide carried byan individual at a biallelic marker.

[0129] The term “mutation” as used herein refers to a difference in DNAsequence between or among different genomes or individuals which has afrequency below 1%.

[0130] The term “haplotype” refers to a combination of alleles presentin an individual or a sample. In the context of the present invention ahaplotype preferably refers to a combination of biallelic marker allelesfound in a given individual and which may be associated with aphenotype.

[0131] The term “polymorphism” as used herein refers to the occurrenceof two or more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism is a single base pairchange. Typically a single nucleotide polymorphism is the replacement ofone nucleotide by another nucleotide at the polymorphic site. Deletionof a single nucleotide or insertion of a single nucleotide, also giverise to single nucleotide polymorphisms. In the context of the presentinvention “single nucleotide polymorphism” preferably refers to a singlenucleotide substitution. Typically, between different genomes or betweendifferent individuals, the polymorphic site may be occupied by twodifferent nucleotides.

[0132] The terms “biallelic polymorphism” and “biallelic marker” areused interchangeably herein to refer to a polymorphism having twoalleles at a fairly high frequency in the population, preferably asingle nucleotide polymorphism. A “biallelic marker allele” refers tothe nucleotide variants present at a biallelic marker site. Typicallythe frequency of the less common allele of the biallelic markers of thepresent invention has been validated to be greater than 1%, preferablythe frequency is greater than 10%, more preferably the frequency is atleast 20% (i.e. heterozygosity rate of at least 0.32), even morepreferably the frequency is at least 30% (i.e. heterozygosity rate of atleast 0.42). A biallelic marker wherein the frequency of the less commonallele is 30% or more is termed a “high quality biallelic marker.”

[0133] The location of nucleotides in a polynucleotide with respect tothe center of the polynucleotide are described herein in the followingmanner. When a polynucleotide has an odd number of nucleotides, thenucleotide at an equal distance from the 3′ and 5′ ends of thepolynucleotide is considered to be “at the center” of thepolynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter”, and so on. For polymorphisms which involve the substitution,insertion or deletion of 1 or more nucleotides, the polymorphism, alleleor biallelic marker is “at the center” of a polynucleotide if thedifference between the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 3′ end of thepolynucleotide, and the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 5′ end of thepolynucleotide is zero or one nucleotide. If this difference is 0 to 3,then the polymorphism is considered to be “within 1 nucleotide of thecenter.” If the difference is 0 to 5, the polymorphism is considered tobe “within 2 nucleotides of the center.” If the difference is 0 to 7,the polymorphism is considered to be “within 3 nucleotides of thecenter,” and so on. For polymorphisms which involve the substitution,insertion or deletion of 1 or more nucleotides, the polymorphism, alleleor biallelic marker is “at the center” of a polynucleotide if thedifference between the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 3′ end of thepolynucleotide, and the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 5′ end of thepolynucleotide is zero or one nucleotide. If this difference is 0 to 3,then the polymorphism is considered to be “within 1 nucleotide of thecenter.” If the difference is 0 to 5, the polymorphism is considered tobe “within 2 nucleotides of the center.” If the difference is 0 to 7,the polymorphism is considered to be “within 3 nucleotides of thecenter,” and so on.

[0134] A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell required to initiate the specific transcription ofa gene.

[0135] As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. More precisely, twoDNA molecules (such as a polynucleotide containing a promoter region anda polynucleotide encoding a desired polypeptide or polynucleotide) aresaid to be “operably linked” if the nature of the linkage between thetwo polynucleotides does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of thepolynucleotide containing the promoter to direct the transcription ofthe coding polynucleotide.

[0136] The term “upstream” is used herein to refer to a location, whichis toward the 5′ end of the polynucleotide from a specific referencepoint.

[0137] The terms “base paired” and “Watson & Crick base paired” are usedinterchangeably herein to refer to nucleotides which can be hydrogenbonded to one another be virtue of their sequence identities in a mannerlike that found in double-helical DNA with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds (See Stryer, L.,Biochemistry, 4^(th) edition, 1995).

[0138] The terms “complementary” or “complement thereof” are used hereinto refer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. This term isapplied to pairs of polynucleotides based solely upon their sequencesand not any particular set of conditions under which the twopolynucleotides would actually bind.

[0139] As used herein, the term “non-human animal” refers to anynon-human vertebrate, birds and more usually mammals, preferablyprimates, farm animals such as swine, goats, sheep, donkeys, and horses,rabbits or rodents, more preferably rats or mice. As used herein, theterm “animal” is used to refer to any vertebrate, preferable a mammal.Both the terms “animal” and “mammal” expressly embrace human subjectsunless preceded with the term “non-human.”

[0140] As used herein, the term “antibody” refers to a polypeptide orgroup of polypeptides which are comprised of at least one bindingdomain, where an antibody binding domain is formed from the folding ofvariable domains of an antibody molecule to form three-dimensionalbinding spaces with an internal surface shape and charge distributioncomplementary to the features of an antigenic determinant of anantigen., which allows an immunological reaction with the antigen.Antibodies include recombinant proteins comprising the binding domains,as wells as fragments, including Fab, Fab′, F(ab)₂, and F(ab′)₂fragments.

[0141] As used herein, an “antigenic determinant” is the portion of anantigen molecule, in this case a 12-LO polypeptide, that determines thespecificity of the antigen-antibody reaction. An “epitope” refers to anantigenic determinant of a polypeptide. An epitope can comprise as fewas 3 amino acids in a spatial conformation which, is unique to theepitope. Generally an epitope consists of at least 6 such amino acids,and more usually at least 8-10 such amino acids. Methods for determiningthe amino acids which make up an epitope include x-ray crystallography,2-dimensional nuclear magnetic resonance, and epitope mapping e.g. thePepscan method described by H. Mario Geysen et al. 1984. Proc. Natl.Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCTPublication No. WO 84/03506, the disclosures of which are incorporatedherein by reference in their entireties.

[0142] As used herein the term “eicosanoid-related biallelic marker”relates to a set of biallelic markers in linkage disequilibrium with allof the genes disclosed in Table 7(A-B) with the exception of FLAP. Allof these genes express proteins that are related to eicosanoidmetabolism. The term eicosanoid-related biallelic marker encompasses allof the biallelic markers disclosed in Table 7(A-B), preferably thebiallelic markers found in SEQ ID Nos. 651-652, 655-724, 726-1072,1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably fromSEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193,and 1195-1300. The preferred eicosanoid-related biallelic marker allelesof the present invention include each one the alleles described inTables 7, 8, 9, and 10 individually or in groups consisting of all thepossible combinations of the alleles included in Tables 7(A-B), 8, 9,and 10, preferably the biallelic markers found in SEQ ID Nos. 1-70,72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos.651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724,726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300.

[0143] As used herein the term “12-LO-related biallelic marker” and“12-lipoxygenase-related biallelic marker” are used interchangeablyherein to relate to all biallelic markers in linkage disequilibrium withthe biallelic markers of the 12-lipoxygenase gene. The term12-LO-related biallelic marker includes both the genic and non-genicbiallelic markers described in Table 2(a-c).

[0144] The term “non-genic” is used herein to describe 12-LO-relatedbiallelic markers, as well as polynucleotides and primers which occuroutside the nucleotide positions shown in the human 12-LO genomicsequence of SEQ ID No. 651. The term “genic” is used herein to describe12-LO-related biallelic markers as well as polynucleotides and primerswhich do occur in the nucleotide positions shown in the human 12-LOgenomic sequence of SEQ ID No 651.

[0145] The term “sequence described in Table 7(A-B)” is used herein torefer to the entire collection of nucleotide sequences or any individualsequence defined in Table 7(A-B). The SEQ ID that contains each“sequence described in Table 7(A-B)” is provided in the column labeled,“SEQ ID NO.” The column labeled “Gene” indicates the gene for which themarker is in closest physical proximity, an indication of whether themarkers have been validated by microsequencing (with a Y indicating thatthe markers have been validated by microsequencing and an N indicatingthat it has not), and an indication of the identity and frequency of theleast common allele determined by genotyping (with a blank left toindicate that the frequency has not yet been reported for some markers).The frequencies were determined from DNA samples collected from a randomUS Caucasian population.

[0146] The term “sequence described in Table 7B” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 7B. The SEQ ID that contains each “sequencedescribed in Table 7B” is provided in the column labeled, “SEQ ID NO.”The column labeled “Gene” indicates the gene for which the marker is inclosest physical proximity, an indication of whether the markers havebeen validated by microsequencing (with a Y indicating that the markershave been validated by microsequencing and an N indicating that it hasnot), and an indication of the identity and frequency of the leastcommon allele determined by genotyping (with a blank left to indicatethat the frequency has not yet been reported for some markers). Thefrequencies were determined from DNA samples collected from a random USCaucasian population. The “Biallelic Marker location in SEQ ID No.”indicates the biallelic marker location within the 47 nucleotidesequence. InTable 7B, this location is 24 for all of the markers.

[0147] The term “sequence described in Table 8” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 8. The SEQ ID that contains each “sequencedescribed in Table 8” is provided in the column labeled, “SEQ ID NO.”The range of nucleotide positions within the Sequence ID of which eachsequence consists is provided in the same row as the Sequence ID in acolumn labeled, “POSITION RANGE OF PREFERRED SEQUENCE”. It should benoted that some of the Sequence ID numbers have multiple sequence rangeslisted, because they contain multiple “sequences described in Table 8.”Unless otherwise noted the term “sequence described in Table 8” is to beconstrued as encompassing sequences that contain either of the twoalleles listed in the columns labeled, “1^(ST) ALLELE” and “2^(ND)ALLELE” at the position identified in field <222> of the allele featurein the appended Sequence Listing for each Sequence ID number referencedin Table 8. For all inventions which relate to biallelic markers orsequences described in Table 8, a preferred set of markers or sequencesexcludes Sequence ID Nos. 1-10, 19, 23-25, and 647-650.

[0148] The term “sequence described in Table 9” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 9. Unless otherwise noted, the “sequencesdescribed in Table 9” consist of the entire sequence of each Sequence IDprovided in the column labeled, “SEQ ID NO.” Also unless otherwise notedthe term “sequence described in Table 9” is to be construed asencompassing sequences that contain either of the two alleles listed inthe columns labeled, “ORIGINAL ALLELE” and “ALTERNATIVE ALLELE” at theposition identified in field <222> of the allele feature in the appendedSequence Listing for each Sequence ID number referenced in Table 9. Forall inventions which relate to biallelic markers or sequences describedin Table 9, a preferred set of markers or sequences excludes Sequence IDNos. 11-18 and 20-21.

[0149] The term “sequence described in Table 10” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 10. Unless otherwise noted, the “sequencesdescribed in Table 10” consist of the entire sequence of each SequenceID provided in the column labeled, “SEQ ID NO.” Also unless otherwisenoted the term “sequence described in Table 10” is to be construed asencompassing sequences that contain either of the two alleles listed inthe columns labeled, “1^(ST) ALLELE” and “2^(ND) ALLELE” at the positionidentified in field <222> of the allele feature in the appended SequenceListing for each Sequence ID number referenced in Table 10. For allinventions which relate to biallelic markers or sequences described inTable 8, a preferred set of markers or sequences excludes Sequence IDNo. 22.

[0150] The term “sequence described in Table 11” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 11. The SEQ ID that contains each “sequencedescribed in Table 11” is provided in the column labeled, “SEQ ID NO.”The range of nucleotide positions within the Sequence ID of which eachsequence consists is provided in the same row as the Sequence ID in acolumn labeled, “POSITION RANGE OF PREFERRED SEQUENCE”. It should benoted that some of the Sequence ID numbers have multiple sequence rangeslisted, because they contain multiple “sequences described in Table 11.”

[0151] The term “sequence described in Table 12” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 12. The SEQ ID that contains each “sequencedescribed in Table 12” is provided in the column labeled “SEQ ID.” Therange of nucleotide positions within the Sequence ID of which half ofthe sequences consists is provided in the same row as the Sequence ID ina column labeled, “POSITION RANGE OF MICROSEQUENCING PRIMERS.” Theremaining half of the sequences described in Table 12 are complementaryto the range of nucleotide positions within the Sequence ID provided inthe same row as the Sequence ID in a column labeled, “COMPLEMENTARYPOSITION RANGE OF MICROSEQUENCING PRIMERS.” For all inventions whichrelate to biallelic markers or sequences described in Table 12, a morepreferred set of markers or sequences consists of those markers orsequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539,541-646, and 651-652.

[0152] The term “sequence described in Table 13” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 13. The SEQ ID that contains each “sequencedescribed in Table 13” is provided in the column labeled, “SEQ ID.” Therange of nucleotide positions within the Sequence ID of which half ofthe sequences consists is provided in the same row as the Sequence ID ina column labeled, “POSITION RANGE OF AMPLIFICATION PRIMERS.” Theremaining half of the sequences described in Table 13 are complementaryto the range of nucleotide positions within the Sequence ID provided inthe same row as the Sequence ID in a column labeled, “COMPLEMENTARYPOSITION RANGE OF AMPLIFICATION PRIMERS.” For all inventions whichrelate to biallelic markers or sequences described in Table 13, a morepreferred set of markers or sequences consists of those markers orsequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539,541-646.

[0153] The term “sequence described in Table 13” is used herein to referto the entire collection of nucleotide sequences or any individualsequence defined in Table 13. The SEQ ID that contains each “sequencedescribed in Table 13” is provided in the column labeled, “SEQ ID”. Therange of nucleotide positions within the Sequence ID of which eachsequence consists is provided in the same row as the Sequence ID in acolumn labeled, “POSITION RANGE OF PROBES”. The sequences which arecomplementary to the ranges listed in the column labeled, “POSITIONRANGE OF PROBES” are also encompassed by the term, “sequence describedin Table 13.” Unless otherwise noted the term “sequence described inTable 13” is to be construed as encompassing sequences that containeither of the two alleles listed in the allele feature in the appendedSequence Listing for each Sequence ID number referenced in Table 13. Forall inventions which relate to biallelic markers or sequences describedin Table 13, a more preferred set of markers or sequences consists ofthose markers or sequences found in SEQ ID Nos. 26-70, 72-418, 425-489,491-530, 532-539, 541-646, and 651-652.

[0154] The terms “biallelic marker described in Table” and “alleledescribed in Table” are used herein to refer to any or all alleles whichare listed in the allele feature in the appended Sequence Listing foreach Sequence ID number referenced in the particular Table beingmentioned.

[0155] The following abbreviations are used in this disclosure: theLTB₄H₂ gene is abbreviated LTB4H2; leukotriene B₄-12-OH dehydrogenase isabbreviated LTB4-12OH; leukotriene B₄ receptor is abbreviated LTB4R;PGD-synthase is abbreviated PGDS; and PG-15-OH dehydrogenase isabbreviated PG15OH.

[0156] Variants and Fragments

[0157] The invention also relates to variants and fragments of thepolynucleotides described herein, particularly of a 12-LO genecontaining one or more biallelic markers according to the invention.

[0158] Variants of polynucleotides, as the term is used herein, arepolynucleotides that differ from a reference polynucleotide. A variantof a polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.Variants of polynucleotides according to the invention include, withoutbeing limited to, nucleotide sequences which are at least 95% identical, preferably at least 99% identical, more particularly at least 99.5%identical, and most preferably at least 99.8% identical to apolynucleotide selected from the group consisting of the polynucleotidesof a sequence from any sequence in the Sequence Listing as well assequences which are complementary thereto or to any polynucleotidefragment of at least 8 consecutive nucleotides of a sequence from anysequence in the Sequence Listing. Nucleotide changes present in avariant polynucleotide may be silent, which means that they do not alterthe amino acids encoded by the polynucleotide. However, nucleotidechanges may also result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. In the context of the present invention,particularly preferred embodiments are those in which thepolynucleotides encode polypeptides which retain substantially the samebiological function or activity as the mature 12-LO protein, or those inwhich the polynucleotides encode polypeptides which maintain or increasea particular biological activity, while reducing a second biologicalactivity. A polynucleotide fragment is a polynucleotide having asequence that is entirely the same as part but not all of a givennucleotide sequence, preferably the nucleotide sequence of a 12-LO gene,and variants thereof. The fragment can be a portion of an exon or of anintron of a 12-LO gene. It can also be a portion of the regulatoryregions of the 12-LO gene preferably of the promoter sequence of the12-LO gene. Such fragments may be “free-standing”, i.e. not part of orfused to other polynucleotides, or they may be comprised within a singlelarger polynucleotide of which they form a part or region. Indeed,several of these fragments may be present within a single largerpolynucleotide.

[0159] Identity Between Nucleic Acids and Polypeptides

[0160] The terms “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Homology is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci.85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol.215(3):403-410,1990; Thompson et al., Nucleic Acids Res.22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402,1996; Altschul et al., Nature Genetics 3:266-272, 1993, the disclosuresof which are incorporated herein by reference in their entireties). In aparticularly preferred embodiment, protein and nucleic acid sequencehomologies are evaluated using the Basic Local Alignment Search Tool(“BLAST”) which is well known in the art (See, e.g., Karlin andAltschul,. Proc. Natl. Acad. Sci. USA 87:2267-2268, 1990; Altschul etal., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., NatureGenetics 3:266-272, 1993; Altschul et al., Nuc. Acids Res. 25:3389-3402,1997, the disclosures of which are incorporated herein by reference intheir entireties). In particular, five specific BLAST programs are usedto perform the following task:

[0161] (1) BLASTP and BLAST3 compare an amino acid query sequenceagainst a protein sequence database;

[0162] (2) BLASTN compares a nucleotide query sequence against anucleotide sequence database;

[0163] (3) BLASTX compares the six-frame conceptual translation productsof a query nucleotide sequence (both strands) against a protein sequencedatabase;

[0164] (4) TBLASTN compares a query protein sequence against anucleotide sequence database translated in all six reading frames (bothstrands); and

[0165] (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database.

[0166] The BLAST programs identify homologous sequences by identifyingsimilar segments, which are referred to herein as “high-scoring segmentpairs,” between a query amino or nucleic acid sequence and a testsequence which is preferably obtained from a protein or nucleic acidsequence database. High-scoring segment pairs are preferably identified(i.e., aligned) by means of a scoring matrix, many of which are known inthe art. Preferably, the scoring matrix used is the BLOSUM62 matrix(Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff,Proteins 17:49-61, 1993, the disclosures of which are incorporatedherein by reference in their entireties). Less preferably, the PAM orPAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds.,Matrices for Detecting Distance Relationships: Atlas of Protein Sequenceand Structure, Washington:National Biomedical Research Foundation, 1978,the disclosure of which is incorporated herein by reference in itsentirety). The BLAST programs evaluate the statistical significance ofall high-scoring segment pairs identified, and preferably selects thosesegments which satisfy a user-specified threshold of significance, suchas a user-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (see, e.g., Karlin andAltschul, Proc. Natl. Acad. Sci. USA 87:2267-2268, 1990, the disclosureof which is incorporated herein by reference in its entirety).

[0167] Stringent Hybridization Conditions

[0168] By way of example and not limitation, procedures using conditionsof high stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 h to overnight at 65° C. in buffercomposed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C., the preferred hybridization temperature,in prehybridization mixture containing 100 μg/ml denatured salmon spermDNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, thehybridization step can be performed at 65° C. in the presence of SSCbuffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals.Following the wash steps, the hybridized probes are detectable byautoradiography. Other conditions of high stringency which may be usedare well known in the art and as cited in Sambrook et al., 1989; andAusubel et al., 1989. These hybridization conditions are suitable for anucleic acid molecule of about 20 nucleotides in length. There is noneed to say that the hybridization conditions described above are to beadapted according to the length of the desired nucleic acid, followingtechniques well-known to one skilled in the art. The suitablehybridization conditions may for example be adapted according to theteachings disclosed in the book of Hames and Higgins (NucleicAcidHybridization: A Practical Approach, IRL Press, Oxford, 1985) or inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), thedisclosures of which are incorporated herein by reference in theirentireties.

[0169] I. Biallelic Markers and Polynucleotides Comprising BiallelicMarkers

[0170] A. Polynucleotides of the Present Invention

[0171] The present invention encompasses polynucleotides for use asprimers and probes in the methods of the invention. Thesepolynucleotides may consist of, consist essentially of, or comprise acontiguous span of nucleotides of a sequence from any sequence in theSequence Listing as well as sequences which are complementary thereto(“complements thereof”). The “contiguous span” may be at least 8, 10,12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000nucleotides in length, to the extent that a contiguous span of theselengths is consistent with the lengths of the particular Sequence ID. Itshould be noted that the polynucleotides of the present invention arenot limited to having the exact flanking sequences surrounding thepolymorphic bases, which are enumerated in the Sequence Listing. Rather,it will be appreciated that the flanking sequences surrounding thebiallelic markers, or any of the primers of probes of the inventionwhich, are more distant from the markers, may be lengthened or shortenedto any extent compatible with their intended use and the presentinvention specifically contemplates such sequences. It will beappreciated that the polynucleotides referred to in the Sequence Listingmay be of any length compatible with their intended use. Also theflanking regions outside of the contiguous span need not be homologousto native flanking sequences which actually occur in human subjects. Theaddition of any nucleotide sequence, which is compatible with thenucleotides intended use is specifically contemplated. The contiguousspan may optionally include the eicosanoid-related biallelic marker insaid sequence. Biallelic markers generally consist of a polymorphism atone single base position. Each biallelic marker therefore corresponds totwo forms of a polynucleotide sequence which, when compared with oneanother, present a nucleotide modification at one position. Usually, thenucleotide modification involves the substitution of one nucleotide foranother. Optionally either the original or the alternative allele of thebiallelic markers disclosed in Table 9, or the first or second alleledisclosed in Tables 8 and 10 may be specified as being present at theeicosanoid-related biallelic marker. Optionally, the biallelic markersmay be specified as 12-214-85, 12-215-272, 12-221-163, 12-225-82,10-234-179, 10-235-272, 10-251-342, 10-395-367, 12-730-58, 12-735-208,12-739-22, 12-540-363, 12-550-206, 10-207-410, 10-171-254, 12-94-110,12-834-290, 10-55-115, 12-857-122, 12-872-175, 12-882-40, 12-888-234,12-278-353, 12-283-386, 12-44-181, 10-343-231, 10-349-216, 10-509-295,10-511-337, 10-349-216, 10-343-231, 10-13-396, 12-570-62, 10-474-320,10-510-173 and 10-342-301 which consist of more complex polymorphismsincluding insertions/deletions of at least one nucleotide. Optionallyeither the original or the alternative allele of these biallelic markersmay be specified as being present at the eicosanoid-related biallelicmarker. Preferred polynucleotides may consist of, consist essentiallyof, or comprise a contiguous span of nucleotides of a sequence from SEQID No 571-595, 600, 606, 613, 620, 628, and 638-639; or more preferablyfrom SEQ ID No 1225-1249, 1254, 1260, 1267, 1274, 1282, 1292 and 1293 aswell as sequences which are complementary thereto. The “contiguous span”may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250,500 or 1000 nucleotides in length, to the extent that a contiguous spanof these lengths is consistent with the lengths of the particularSequence ID. The contiguous span may optionally comprise a biallelicmarker selected from the group consisting of biallelic markers12-214-85, 12-215-272, 12-221-163, 12-225-82, 10-234-179, 10-235-272,10-251-342, 10-395-367, 12-730-58, 12-735-208, 12-739-22, 12-540-363,12-550-206, 10-207-410, 10-171-254, 12-94-110, 12-834-290, 10-55-115,12-857-122, 12-872-175, 12-882-40, 12-888-234, 12-278-353, 12-283-386,12-44-181, 10-343-231, 10-349-216, 10-509-295, 10-511-337, 10-349-216,10-343-231, 10-13-396, 12-570-62, 10-474-320, 10-510-173 and 10-342-301.

[0172] The invention also relates to polynucleotides that hybridize,under conditions of high or intermediate stringency, to a polynucleotideof a sequence from any sequence in the Sequence Listing as well assequences, which are complementary thereto. Preferably suchpolynucleotides are at least 20, 25, 35, 40, 50, 70, 80, 100, 250, 500or 1000 nucleotides in length, to the extent that a polynucleotide ofthese lengths is consistent with the lengths of the particular SequenceID. Preferred polynucleotides comprise an eicosanoid-related biallelicmarker. Optionally either the original or the alternative allele of thebiallelic markers disclosed in Table 10 may be specified as beingpresent at the eicosanoid-related biallelic marker. Conditions of highand intermediate stringency are further described in III.C.4 “Methods ofGenotyping DNA Samples for Biallelic Markers-Hybridization assaymethods.”

[0173] The preferred polynucleotides of the invention include thesequence ranges included in any one the sequence ranges of Tables 8, 11,and 14 individually or in groups consisting of all the possiblecombinations of the ranges of included in Tables 8, 11, and 14. Thepreferred polynucleotides of the invention also include fragments of atleast 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or1000 consecutive nucleotides of the sequence ranges included in any oneof the sequence ranges of Tables 8, 11, and 14 to the extent thatfragments of these lengths are consistent with the lengths of theparticular sequence range. The preferred polynucleotides of theinvention also include fragments of at least 8, 10, 12, 15, 18, 20, 25,35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides of thesequence complementary to the sequence ranges included in any one of thesequence ranges of Tables 8, 11, and 14 to the extent that fragments ofthese lengths are consistent with the lengths of the particular sequencerange.

[0174] Particularly preferred polynucleotides of the invention includeisolated, purified or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 651,wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 ofthe following nucleotide positions of SEQ ID No. 651:1 to 2584, 4425 to5551, 5634 to 5757, 5881 to 5995, 6100 to 6348, 6510 to 7378, 7523 to8644, 8855 to 12253, 12341 to12853, 13024 to 13307, 13430 to 16566,16668 to 16774, 16946 to 17062, 17555 to 20674; and the complementsthereof. Other particularly preferred polynucleotides of the inventioninclude isolated, purified or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQID No. 651 and the complements thereof; wherein said contiguous spancomprises at least one nucleotide positions selected from the groupconsisting of: a C at position 3355, a G at position 3488, a G atposition 3489, and a G at position 3708 of SEQ ID No. 651.

[0175] Additional preferred polynucleotides of the invention includeisolated, purified or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQID No. 652, wherein said contiguous span comprises a T at position 1205of SEQ ID No. 652 or nucleotide positions 2151 to 2157of SEQ ID No. 652;and the complements thereof.

[0176] The present invention further embodies isolated, purified, andrecombinant polynucleotides which encode polypeptides comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of SEQ ID No. 653, wherein said contiguous span comprises atleast one amino acid position selected from the group consisting of thefollowing: an His residue et amino acid position 189, an His residue atamino acid position 225, a Cys residue at amino acid position 243, anArg residue at amino acid position 261, an Asn residue at amino acidposition 322, an Arg residue at amino acid position 337, a Asn residueat amino acid position 362, an Asn at amino acid position 568 and a Lysresidue at amino acid position 574. The present invention furtherprovides isolated, purified, and recombinant polynucleotides whichencode polypeptides comprising a contiguous span of at least 6 aminoacids, preferably at least 8 to 10 amino acids, more preferably at least12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653,wherein said contiguous span comprises at least one of amino acidpositions 110-131 of SEQ ID No. 653.

[0177] Particularly preferred polynucleotides of the present inventioninclude purified, isolated or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence selectedfrom the group consisting of SEQ ID Nos. 26-68, 614-646, and 651-652; ormore preferably from SEQ ID No 651-652, 680-722, and 1268-1300, or thecomplements thereof, wherein said span includes a12-lipoxygenase-related biallelic marker. Optionally said biallelicmarker is selected from the biallelic markers described in Table 2(a-c)and even more preferably said biallelic marker is selected frombiallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366,10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97,10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230,and 12-223-207. Optionally either allele of the biallelic markersdescribed above in the definition of 12-lipoxygenase-related biallelicmarker is specified as being present at the 12-lipoxygenase-relatedbiallelic marker.

[0178] Particularly preferred polynucleotides of the present inventioninclude purified, isolated or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ IDNo. 651 and the complements thereof; wherein said contiguous spancomprises a least one nucleotide positions selected from the groupconsisting of: a T at position 2323, a C at position 2341, an A atposition 2623, an A at position 2832, a C at position 2844, an A atposition 2934, an A at position 2947, a G at position 3802, a G atposition 4062, a C at position 4088, a T at position 4109, a T atposition 4170, an A at position 6019, a C at position 6375, a C atposition 6429, an A at position 6467, a G at position 6484, an A atposition 8658, a G at position 8703, an A at position 8777, a G atposition 8785, a G at position 13341, an A at position 16836, an A atposition 16854, and a T at position 17355 of SEQ ID No. 651.

[0179] Particularly preferred polynucleotides of the present inventioninclude purified, isolated or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ IDNo. 652 and the complements thereof; wherein said contiguous spancomprises a least one nucleotide position selected from the groupconsisting of: G at position 366, an A at position 605, a C at position712, a T at position 766, an A at position 804, a G at position 821, anA at position 1004, a G at position 1049, an A at position 1123, a G atposition 1131, a G at position 1491, an A at position 1742, an A atposition 1760, an A at position 1941, and a T at position 2144 of SEQ IDNo. 652.

[0180] Table 2(a-c) contains a list of preferred 12-LO-related biallelicmarkers. Each marker is described by indicating its Marker ID, theposition of the marker in the SEQ ID and the two most common alleles.TABLE 2a NON-GENOMIC BIALLELIC MARKERS POSITION OF POSITION OF BIALLELICBIALLELIC MARKER IN SEQ MARKER IN SEQ ID (FIG. 2A) ID (FIG. 2B)BIALLELIC SEQ ID SEQ ID MARKER ID ALLELES No. Position No Position12-196-119 C/T 44 119 698 24 12-197-244 C/T 45 243 699 24 12-198-128 A/G46 128 700 24 12-208-35 A/T 48 35 702 24 12-214-129 C/T 49 129 703 2412-214-151 G/C 50 151 704 24 12-214-360 C/G 51 358 705 24 12-214-85Deletion 571 85 1225 24 CCTAT 12-215-272 Deletion T 572 271 1226 2412-215-467 G/T 52 466 706 24 12-216-421 A/G 53 418 707 24 12-219-230 A/G54 229 708 24 12-219-256 C/T 55 255 709 24 12-221-163 GTCCTA/T 573 1631227 24 12-221-302 A/C 57 302 711 24 12-223-179 A/G 58 179 712 2412-223-207 C/T 59 207 713 24 12-225-541 C/T 60 540 714 24 12-225-82Deletion T 574 82 1228 24 12-226-167 C/G 61 166 715 24 12-226-458 C/T 62455 716 24 12-229-332 G/C 63 332 717 24 12-229-351 G/C 64 351 718 2412-230-364 C/T 65 364 719 24 12-231-100 C/T 66 99 720 24 12-231-148 C/T67 147 721 24 12-231-266 C/T 68 265 722 24

[0181] TABLE 2b BIALLELIC MARKERS IN GENOMIC SEQUENCE (SEQ ID No. 651)BIALLELIC POSITION OF BIALLELIC MARKER ID ALLELES MARKER IN SEQ ID10-508-191 C/T 1128 10-508-245 C/T 1182 10-510-173 ATTTA/TTTTTT 182710-511-62 C/T 2048 10-511-337 Insertion of T 2323 10-512-36 G/C 234110-512-318 A/G 2623 10-513-250 A/G 2832 10-513-262 C/T 2844 10-513-352A/G 2934 10-513-365 A/G 2947 12-206-81 A/G 3802 10-343-231 Deletion of C4062 12-206-366 C/T 4088 10-343-278 C/T 4109 10-343-339 G/T 417010-346-23 A/G 5903 10-346-141 A/G 6019 10-346-263 G/C 6141 10-346-305C/T 6183 10-347-74 A/G 6338 10-347-111 G/C 6375 10-347-165 C/T 642910-347-203 A/G 6467 10-347-220 A/G 6484 10-347-271 A/T 6534 10-347-348A/G 6611 10-348-391 A/G 7668 10-349-47 C/T 8608 10-349-97 A/G 865810-349-142 G/C 8703 10-349-216 Deletion of CTG 8777 10-349-224 G/T 878510-349-368 C/T 8926 10-350-72 C/T 12171 10-350-332 C/T 12429 10-507-170A/G 13341 10-507-321 A/C 13492 10-507-353 C/T 13524 10-507-364 C/T 1353510-507-405 C/T 13576 12-220-48 G/A 15194 10-339-32 C/T 16468 10-339-124C/T 16559 10-340-112 A/C 16836 10-340-130 A/T 16854 10-340-238 A/G 1696210-341-116 A/G 17152 10-341-319 C/T 17355 10-342-301 Insertion of A17623 10-342-373 C/T 17695

[0182] TABLE 2c BIALLELIC MARKERS IN 12-LO cDNA (SEQ ID No 652) POSITIONOF BIALLELIC MARKER BIALLELIC MARKER ID ALLELES IN SEQ ID 10-343-231Deletion of C 366 10-346-141 A/G 605 10-347-111 G/C 712 10-347-165 C/T766 10-347-203 A/G 804 10-347-220 A/G 821 10-349-142 G/C 1049 10-349-216Deletion of CTG 1123 10-349-224 G/T 1131 10-507-170 A/G 1491 10-340-112A/C 1742 10-340-130 A/T 1760 10-341-116 A/G 1941 10-341-319 C/T 2144

[0183] The primers of the present invention may be designed from thedisclosed sequences for any method known in the art. A preferred set ofprimers is fashioned such that the 3′ end of the contiguous span ofidentity with the sequences of the Sequence Listing is present at the 3′end of the primer. Such a configuration allows the 3′ end of the primerto hybridize to a selected nucleic acid sequence and dramaticallyincreases the efficiency of the primer for amplification or sequencingreactions. In a preferred set of primers the contiguous span is found inone of the sequences described in Table 11. Allele specific primers maybe designed such that a biallelic marker is at the 3′ end of thecontiguous span and the contiguous span is present at the 3′ end of theprimer. Such allele specific primers tend to selectively prime anamplification or sequencing reaction so long as they are used with anucleic acid sample that contains one of the two alleles present at abiallelic marker. The 3′ end of primers of the invention may be locatedwithin or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250,500, or 1000, to the extent that this distance is consistent with theparticular Sequence ID, nucleotides upstream of an eicosanoid-relatedbiallelic marker in said sequence or at any other location which isappropriate for their intended use in sequencing, amplification or thelocation of novel sequences or markers. A list of preferredamplification primers is disclosed in Table 13. A more preferred set ofamplification primers is described in Table 13 in SEQ ID Nos. 26-70,72-418, 425-489, 491-530, 532-539, 541-646, and 651-652. Primers withtheir 3′ ends located 1 nucleotide upstream of an eicosanoid-relatedbiallelic marker have a special utility as microsequencing assays.Preferred microsequencing primers are described in Table 12. A morepreferred set of microsequencing primers is described in Table 12 in SEQID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.

[0184] The probes of the present invention may be designed from thedisclosed sequences for any method known in the art, particularlymethods which allow for testing if a particular sequence or markerdisclosed herein is present. A preferred set of probes may be designedfor use in the hybridization assays of the invention in any manner knownin the art such that they selectively bind to one allele of a biallelicmarker, but not the other under any particular set of assay conditions.Preferred hybridization probes may consists of, consist essentially of,or comprise a contiguous span which ranges in length from 8, 10, 12, 15,18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specifiedas being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length andincluding an eicosanoid-related biallelic marker of said sequence.Optionally the original allele or alternative allele disclosed in Tables9 and 10 may be specified as being present at the biallelic marker site.Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1nucleotides of the center of the hybridization probe or at the center ofsaid probe. A particularly preferred set of hybridization probes isdisclosed in Table 14 or a sequence complementary thereto.

[0185] Any of the polynucleotides of the present invention can belabeled, if desired, by incorporating a label detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include radioactive substances,fluorescent dyes or biotin. Preferably, polynucleotides are labeled attheir 3′ and 5′ ends. A label can also be used to capture the primer, soas to facilitate the immobilization of either the primer or a primerextension product, such as amplified DNA, on a solid support. A capturelabel is attached to the primers or probes and can be a specific bindingmember which forms a binding pair with the solid's phase reagent'sspecific binding member (e.g. biotin and streptavidin). Thereforedepending upon the type of label carried by a polynucleotide or a probe,it may be employed to capture or to detect the target DNA. Further, itwill be understood that the polynucleotides, primers or probes providedherein, may, themselves, serve as the capture label. For example, in thecase where a solid phase reagent's binding member is a nucleic acidsequence, it may be selected such that it binds a complementary portionof a primer or probe to thereby immobilize the primer or probe to thesolid phase. In cases where a polynucleotide probe itself serves as thebinding member, those skilled in the art will recognize that the probewill contain a sequence or “tail” that is not complementary to thetarget. In the case where a polynucleotide primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase. DNA Labeling techniquesare well known to the skilled technician.

[0186] Any of the polynucleotides, primers and probes of the presentinvention can be conveniently immobilized on a solid support. Solidsupports are known to those skilled in the art and include the walls ofwells of a reaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, duracytes® andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleic acids on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microtiter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes® andother configurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the inventions to a single solidsupport. In addition, polynucleotides other than those of the inventionmay be attached to the same solid support as one or more polynucleotidesof the invention.

[0187] Any polynucleotide provided herein may be attached in overlappingareas or at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092. These arrays may generally be produced using mechanicalsynthesis methods or light directed synthesis methods, which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis (Fodor et al., Science, 251:767-777, 1991).The immobilization of arrays of oligonucleotides on solid supports hasbeen rendered possible by the development of a technology generallyidentified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™)in which, typically, probes are immobilized in a high density array on asolid surface of a chip. Examples of VLSIPS™ technologies are providedin U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO90/15070, WO 92/10092 and WO 95/11995, which describe methods forforming oligonucleotide arrays through techniques such as light-directedsynthesis techniques. In designing strategies aimed at providing arraysof nucleotides immobilized on solid supports, further presentationstrategies were developed to order and display the oligonucleotidearrays on the chips in an attempt to maximize hybridization patterns andsequence information. Examples of such presentation strategies aredisclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 andWO 97/31256.

[0188] Oligonucleotide arrays may comprise at least one of the sequencesselected from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489,491-530, 532-539, and 541-652, and the complements thereof; preferablySEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193,and 1195-1304, and the complements thereof; or more preferably from SEQID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300, and the complements thereof or a fragment thereof of at least8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000consecutive nucleotides, to the extent that fragments of these lengthsis consistent with the lengths of the particular Sequence ID, fordetermining whether a sample contains one or more alleles of thebiallelic markers of the present invention. Oligonucleotide arrays mayalso comprise at least one of the sequences selected from the groupconsisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and541-652, and the complements thereof; preferably SEQ ID Nos. 651-652,655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, andthe complements thereof, or more preferably from SEQ ID Nos. 651-652,680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, andthe complements thereof or a fragment thereof of at least 8, 10, 12, 15,18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutivenucleotides, to the extent that fragments of these lengths is consistentwith the lengths of the particular Sequence ID, for amplifying one ormore alleles of the biallelic markers of Table 7(A-B). In otherembodiments, arrays may also comprise at least one of the sequencesselected from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489,491-530, 532-539, and 541-652, and the complements thereof; preferablySEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193,and 1195-1304, and the complements thereof; or more preferably from SEQID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1300, and the complements thereof or a fragment thereof of at least8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000consecutive nucleotides, to the extent that fragments of these lengthsis consistent with the lengths of the particular Sequence ID, forconducting microsequencing analyses to determine whether a samplecontains one or more alleles of the biallelic markers of the invention.In still further embodiments, the oligonucleotide array may comprise atleast one of the sequences selecting from the group consisting of SEQ IDNos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652, ormore preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143,1145-1184, 1186-1193, and 1195-1300; and the sequences complementarythereto or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35,40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to theextent that fragments of these lengths is consistent with the lengths ofthe particular Sequence ID, for determining whether a sample containsone or more alleles of the biallelic markers of the present invention.In still further embodiments, the oligonucleotide array may comprise atleast one of the novel sequences listed in the fifth column of Table 8or the sequences complementary thereto or a fragment comprising at least8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000consecutive nucleotides thereof to the extent that fragments of theselengths are consistent with the lengths of the particular novelsequences.

[0189] The present invention also encompasses diagnostic kits comprisingone or more polynucleotides of the invention, optionally with a portionor all of the necessary reagents and instructions for genotyping a testsubject by determining the identity of a nucleotide at aneicosanoid-related biallelic marker. The polynucleotides of a kit mayoptionally be attached to a solid support, or be part of an array oraddressable array of polynucleotides. The kit may provide for thedetermination of the identity of the nucleotide at a marker position byany method known in the art including, but not limited to, a sequencingassay method, a microsequencing assay method, a hybridization assaymethod, an allele specific amplification method, or a mismatch detectionassay based on polymerases and/or ligases. Optionally such a kit mayinclude instructions for scoring the results of the determination withrespect to the test subjects' risk of contracting a diseases involvingarachidonic acid metabolism, or likely response to an agent acting onarachidonic acid metabolism, or chances of suffering from side effectsto an agent acting on arachidonic acid metabolism. Preferably such a kitmay include instructions for scoring the results of the determinationwith respect to the subjects risk of developing hepatotoxicity upontreatment with the anti-asthmatic drug zileuton.

[0190] B. Genomic Sequences of the 12-LO Gene and Biallelic Markers

[0191] The present invention encompasses the genomic sequence of the12-LO gene of SEQ ID No. 651. The 12-LO genomic sequences comprise exonsand introns. Particularly preferred genomic sequences of the 12-LO geneinclude isolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 651,wherein said contiguous span comprises at least 1 one of the followingnucleotide positions of SEQ ID No. 651: 1 to 2584,4425 to 5551, 5634 to5757, 5881 to 5995, 6100 to 6348, 6510 to 7378, 7523 to 8644, 8855 to12253, 12341 to12853, 13024 to 13307, 13430 to 16566, 16668 to 16774,16946 to 17062, 17555 to 20674; and the complements thereof. The nucleicacids defining the 12-LO intronic polynucleotides may be used asoligonucleotide primers or probes in order to detect the presence of acopy of the 12-LO gene in a test sample, or alternatively in order toamplify a target nucleotide sequence within the12-LO sequences. Otherparticularly preferred genomic sequences of the invention includeisolated, purified or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQID No. 651 and the complements thereof; wherein said contiguous spancomprises at least one nucleotide positions selected from the groupconsisting of: a C at position 3355, a G at position 3488, a G atposition 3489, and a G at position 3708 of SEQ ID No. 651.

[0192] The present invention further provides 12-lipoxygenase intron andexon polynucleotide sequences including biallelic markers. Particularlypreferred polynucleotides of the present invention include purified,isolated or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 651 or thecomplements thereof, wherein said span includes a12-lipoxygenase-related biallelic marker. Optionally said biallelicmarker is selected from the biallelic markers described in Table 2(a-c)and even more preferably said biallelic marker is selected frombiallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366,10-346-141, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220,10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421,12-219-230, and 12-223-207. Particularly preferred genomic sequences ofthe present invention include purified, isolated or recombinantpolynucleotides comprising a contiguous span of at least 12, 15, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of a sequence of SEQ ID No. 651 and the complements thereof,wherein said contiguous span comprises a least one nucleotide positionsselected from the group consisting of: a T at position 2323, a C atposition 2341, an A at position 2623, an A at position 2832, a C atposition 2844, an A at position 2934, an A at position 2947, a G atposition 3802, a G at position 4062, a C at position 4088, a T atposition 4109, a T at position 4170, an A at position 6019, a C atposition 6375, a C at position 6429, an A at position 6467, a G atposition 6484, an A at position 8658, a G at position 8703, an A atposition 8777, a G at position 8785, a G at position 13341, an A atposition 16836, an A at position 16854, and a T at position 17355 of SEQID No. 651.

[0193] The genomic sequence of the 12-LO gene contains regulatorysequences both in the non-coding 5′-flanking region and in thenon-coding 3′-flanking region that border the 12-LO transcribed regioncontaining the 14 exons of this gene. 5′-regulatory sequences of the12-LO gene comprise the polynucleotide sequences located between thenucleotide in position 1 and the nucleotide in position 3124 of thenucleotide sequence of SEQ ID No. 651, more preferably between positions1 and 2195 of SEQ ID No. 651. 3′-regulatory sequences of the 12-LO genecomprise the polynucleotide sequences located between the nucleotide inposition 17555 and the nucleotide in position 20674 of the nucleotidesequence of SEQ ID No. 651.

[0194] The promoter activity of the regulatory regions contained in the12-LO gene of polynucleotide sequence of SEQ ID No. 651 can be assessedby any known method. Methods for identifying the polynucleotidefragments of SEQ ID No. 651 involved in the regulation of the expressionof the 12-LO gene are well-known to those skilled in the art (seeSambrook et al., Molecular Cloning A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Anexample of a typical method, that can be used, involves a recombinantvector carrying a reporter gene and genomic sequences from the 12-LOgenomic sequence of SEQ ID No. 651. Briefly, the expression of thereporter gene (for example beta galactosidase or chloramphenicol acetyltransferase) is detected when placed under the control of a biologicallyactive polynucleotide fragment. Genomic sequences located upstream ofthe first exon of the 12-LO gene may be cloned into any suitablepromoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer,pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectorsavailable from Clontech, or pGL2-basic or pGL3-basic promoterlessluciferase reporter gene vector from Promega. Each of these promoterreporter vectors include multiple cloning sites positioned upstream of areporter gene encoding a readily assayable protein such as secretedalkaline phosphatase, luciferase, beta galactosidase, or greenfluorescent protein. The sequences upstream the first 12-LO exon areinserted into the cloning sites upstream of the reporter gene in bothorientations and introduced into an appropriate host cell. The level ofreporter protein is assayed and compared to the level obtained with avector lacking an insert in the cloning site. The presence of anelevated expression level in the vector containing the insert withrespect to the control vector indicates the presence of a promoter inthe insert.

[0195] Promoter sequences within the 5′ non-coding regions of the 12-LOgene may be further defined by constructing nested 5′ and/or 3′deletions using conventional techniques such as Exonuclease III orappropriate restriction endonuclease digestion. The resulting deletionfragments can be inserted into the promoter reporter vector to determinewhether the deletion has reduced or obliterated promoter activity, suchas described, for example, by Coles et al. (Hum. Mol. Genet., 7:791-800,1998, the disclosure of which is incorporated herein by reference in itsentirety). In this way, the boundaries of the promoters may be defined.If desired, potential individual regulatory sites within the promotermay be identified using site directed mutagenesis or linker scanning toobliterate potential transcription factor binding sites within thepromoter individually or in combination. The effects of these mutationson transcription levels may be determined by inserting the mutationsinto cloning sites in promoter reporter vectors. This type of assays arewell known to those skilled in the art and are further described in WO97/17359, U.S. Pat. No. 5,374,544, EP 582 796, U.S. Pat. No. 5,698,389,U.S. Pat. No. 5,643,746, U.S. Pat. No. 5,502,176, and U.S. Pat. No.5,266,488, the disclosures of which are incorporated herein by referencein their entireties.

[0196] The activity and the specificity of the promoter of the 12-LOgene can further be assessed by monitoring the expression level of adetectable polynucleotide operably linked to the 12-LO promoter indifferent types of cells and tissues. The detectable polynucleotide maybe either a polynucleotide that specifically hybridizes with apredefined oligonucleotide probe, or a polynucleotide encoding adetectable protein, including a 12-LO polypeptide or a fragment or avariant thereof. This type of assay is well known to those skilled inthe art and is described in U.S. Pat. No. 5,502,176, and U.S. Pat. No.5,266,488, the disclosures of which are incorporated herein by referencein their entireties.

[0197] Polynucleotides carrying the regulatory elements located both atthe 5′ end and at the 3′ end of the 12-LO coding region may beadvantageously used to control the transcriptional and translationalactivity of a heterologous polynucleotide of interest, saidpolynucleotide being heterologous as regards to the 12-LO regulatoryregion.

[0198] Thus, the present invention also concerns a purified, isolated,and recombinant nucleic acid comprising a polynucleotide which, isselected from the group consisting of, the polynucleotide sequenceslocated between the nucleotide in position 1 and the nucleotide inposition 3124 of the nucleotide sequence of SEQ ID No. 651, morepreferably between positions 1 and 2195 of SEQ ID No. 651 and thepolynucleotide sequences located between the nucleotide in position17555 and the nucleotide in position 20674 of SEQ ID No. 651; or asequence complementary thereto or a biologically active fragmentthereof.

[0199] A “biologically active” fragment of SEQ ID No. 651 according tothe present invention is a polynucleotide comprising or alternativelyconsisting of a fragment of said polynucleotide which is functional as aregulatory region for expressing a recombinant polypeptide or arecombinant polynucleotide in a recombinant cell host.

[0200] For the purpose of the invention, a nucleic acid orpolynucleotide is “functional” as a regulatory region for expressing arecombinant polypeptide or a recombinant polynucleotide if saidregulatory polynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide.

[0201] The regulatory polynucleotides according to the invention may beadvantageously part of a recombinant expression vector that may be usedto express a coding sequence in a desired host cell or host organism.

[0202] A further object of the invention consists of an isolatedpolynucleotide comprising:

[0203] a) a nucleic acid comprising a regulatory nucleotide sequenceselected from the group consisting of a nucleotide sequence comprising apolynucleotide of SEQ ID No. 651;

[0204] b) a polynucleotide encoding a desired polypeptide or a nucleicacid of interest, operably linked to the nucleic acid defined in (a)above.

[0205] The polypeptide encoded by the nucleic acid described above maybe of various nature or origin, encompassing proteins of prokaryotic oreukaryotic origin. Among the polypeptides expressed under the control ofa 12-LO regulatory region, there may be cited bacterial, fungal or viralantigens. Also encompassed are eukaryotic proteins such as intracellularproteins, for example “house keeping” proteins, membrane-bound proteins,for example receptors, and secreted proteins, for example cytokines. Ina specific embodiment, the desired polypeptide may be the 12-LO protein,especially the protein of the amino acid sequence of SEQ ID No. 653 and654.

[0206] The desired nucleic acids encoded by the above describedpolynucleotide, usually a RNA molecule, may be complementary to adesired coding polynucleotide, for example to the 12-LO coding sequence,and thus useful as an antisense polynucleotide.

[0207] Such a polynucleotide may be included in a recombinant expressionvector in order to express the desired polypeptide or the desirednucleic acid in host cell or in a host organism.

[0208] C. cDNA Sequences of the 12-LO Gene and Biallelic Markers

[0209] The present invention provides a 12-lipoxygenase cDNA of SEQ IDNo. 652. The Open Reading Frame encoding the 12-LO protein spans fromthe nucleotide in position 40 to the nucleotide in position 2028 of thepolynucleotide sequence of SEQ ID No. 652. The cDNA of SEQ ID No. 652also includes a 5′-UTR region (1-40) and a 3′-UTR (2028-2343) region.

[0210] Additional preferred cDNA polynucleotides of the inventioninclude isolated, purified or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQID No. 652 and the complements thereof. Additional preferredpolynucleotides include isolated, purified or recombinantpolynucleotides comprising a contiguous span of at least 12, 15, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides from a sequence of SEQ ID No. 652, wherein said contiguousspan comprises a T at position 1205 of SEQ ID No. 652 or nucleotidepositions 2151 to 2157of SEQ ID No. 652; and the complements thereof.

[0211] Preferred cDNA fragments comprise a biallelic marker selectedfrom the group consisting of 10-343-231, 10-346-141, 10-347-111,10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-142, 10-349-216,10-349-224, 10-507-170, 10-340-112, 10-340-130, 10-341-116 and10-341-319. Some biallelic polymorphisms represent silent nucleotidesubstitutions but biallelic markers 10-346-141, 10-347-111, 10-347-165,10-347-220, 10-349-97, 10-349-142, 10-349-216, 10-340-112, 10-340-130are associated with amino acid changes in the corresponding12-lipoxygenase polypeptide. One allele of biallelic marker 10-343-231(polymorphic deletion of a C nucleotide at position 366 of SEQ ID No.652) causes a frame shift in the open reading frame of the 12-LO cDNA ofSEQ ID No. 652 resulting in the novel polypeptide of SEQ ID No. 653.12-LO polypeptides of SEQ ID Nos. 653 and 654 of the present inventionare further described below.

[0212] Other preferred cDNA fragments comprise a contiguous span of atleast 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides from a sequence of SEQ ID No. 652, wherein saidcontiguous span comprises a T at position 1205 of SEQ ID No. 652; andthe complements thereof. 12-LO cDNA fragments comprise a contiguous spanof at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652,wherein said contiguous span comprises a T at position 1205 of SEQ IDNo. 652 encode novel 12-LO polypeptides of SEQ ID No. 653 comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of SEQ ID No. 653, wherein said contiguous span comprises aLeu residue at amino acid position 389 of SEQ ID No. 653.

[0213] The polynucleotide disclosed above that contains the codingsequence of the 12-LO gene of the invention may be expressed in adesired host cell or a desired host organism, when this polynucleotideis placed under the control of suitable expression signals. Theexpression signals may be either the expression signals contained in theregulatory regions in the 12-LO gene of the invention or may beexogenous regulatory nucleic sequences. Such a polynucleotide, whenplaced under the suitable expression signals, may also be inserted in avector for its expression.

[0214] Another preferred cDNA fragment comprises the 5′-UTR(5′regulatory sequence) region beginning at position 1 and ending atposition 39 of SEQ ID No. 652. Another preferred cDNA fragment comprisesthe 3′-UTR (3′regulatory sequence) region beginning at position 2029 andending at position 2343 of SEQ ID No. 652. Preferably said 3′-UTR regioncomprises biallelic marker 10-341-319 or nucleotide positions 2151 to2157of SEQ ID No. 652.

[0215] D. Polynucleotide Constructs, Recombinant Vectors, Host Cells andTransgenic Animals

[0216] The terms “polynucleotide construct” and “recombinantpolynucleotide” are used interchangeably herein to refer to linear orcircular, purified or isolated polynucleotides that have beenartificially designed and which comprise at least two nucleotidesequences that are not found as contiguous nucleotide sequences in theirinitial natural environment.

[0217] Polynucleotide Constructs

[0218] 1. DNA Constructs for Expressing the 12-LO Gene in RecombinantHost Cells and in Transgenic Animals.

[0219] In order to study the physiological and phenotype consequences ofa lack of synthesis of the 12-LO protein, both at the cellular level andat the multicellular organism level, in particular as regards todisorders related to abnormal cell proliferation, notably cancers, theinvention also encompasses DNA constructs and recombinant vectorsenabling a conditional expression of a specific allele of the 12-LOgenomic sequence or cDNA

[0220] A first preferred DNA construct is based on the tetracyclineresistance operon tet from E. coli transposon Tn110 for controllingthe12-LO gene expression, such as described by Gossen et al. (Science,268:1766-1769, 1995, the disclosure of which is incorporated herein byreference in its entirety). Such a DNA construct contains seven tetoperator sequences from Tn10 (tetop) that are fused to either a minimalpromoter or a 5′-regulatory sequence of the 12-LO gene, said minimalpromoter or said 12-LO regulatory sequence being operably linked to apolynucleotide of interest that codes either for a sense or an antisenseoligonucleotide or for a polypeptide, including a 12-LO polypeptide or apeptide fragment thereof. This DNA construct is functional as aconditional expression system for the nucleotide sequence of interestwhen the same cell also comprises a nucleotide sequence coding foreither the wild type (tTA) or the mutant (rTA) repressor fused to theactivating domain of viral protein VP16 of herpes simplex virus, placedunder the control of a promoter, such as the HCMVIE1 enhancer/promoteror the MMTV-LTR. Indeed, a preferred DNA construct of the invention willcomprise both the polynucleotide containing the tet operator sequencesand the polynucleotide containing a sequence coding for the tTA or therTA repressor. In the specific embodiment wherein the conditionalexpression DNA construct contains the sequence encoding the mutanttetracycline repressor rTA, the expression of the polynucleotide ofinterest is silent in the absence of tetracycline and induced in itspresence.

[0221] 2. DNA Constructs Allowing Homologous Recombination: ReplacementVectors.

[0222] A second preferred DNA construct will comprise, from 5′-end to3′-end: (a) a first nucleotide sequence that is comprised in the 12-LOgenomic sequence; (b) a nucleotide sequence comprising a positiveselection marker, such as the marker for neomycine resistance (neo); and(c) a second nucleotide sequence that is comprised in the 12-LO genomicsequence, and is located on the genome downstream the first 12-LOnucleotide sequence (a).

[0223] In a preferred embodiment, this DNA construct also comprises anegative selection marker located upstream the nucleotide sequence (a)or downstream the nucleotide sequence (c). Preferably, the negativeselection marker consists of the thymidine kinase (tk) gene (Thomas etal., Cell, 44:419-428, 1986, the disclosure of which is incorporatedherein by reference in its entirety), the hygromycine beta gene (TeRiele et al., Nature, 348:649-651, 1990, the disclosure of which isincorporated herein by reference in its entirety), the hprt gene (Vander Lugt et al., Gene, 105:263-267, 1991; Reid et al., Proc. Natl. Acad.Sci. USA, 87:4299-4303, 1990, the disclosures of which are incorporatedherein by reference in their entireties) or the Diphteria toxin Afragment (Dt-A) gene (Nada et al., Cell, 73:1125-1135, 1993; Yagi etal., Proc. Natl; Acad. Sci. USA, 87:9918-9922, 1990, the disclosures ofwhich are incorporated herein by reference in their entireties).Preferably, the positive selection marker is located within a 12-LO exonsequence so as to interrupt the sequence encoding a 12-LO protein.

[0224] These replacement vectors are further described by Mansour et al.(Nature, 336:348-352, 1988, the disclosure of which is incorporatedherein by reference in its entirety) and Koller et al. (Ann. Rev.Immunol., 10:705-730, 1992, the disclosure of which is incorporatedherein by reference in its entirety).

[0225] The first and second nucleotide sequences (a) and (c) may beindifferently located within a 12-LO regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) is ranging from 1 to 50 kb, preferably from 1 to10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4kb.

[0226] 3. DNA Constructs Allowing Homologous Recombination: Cre-loxPSystem.

[0227] These new DNA constructs make use of the site specificrecombination system of the P1 phage. The P1 phage possesses arecombinase called Cre which, interacts specifically with a 34 basepairs loxP site. The loxP site is composed of two palindromic sequencesof 13 bp separated by a 8 bp conserved sequence (Hoess et al., NucleicAcids Res., 14:2287-2300, 1986, the disclosure of which is incorporatedherein by reference in its entirety). The recombination by the Creenzyme between two loxP sites having an identical orientation leads tothe deletion of the DNA fragment.

[0228] The Cre-loxP system used in combination with a homologousrecombination technique was first described by Gu et al. (Cell,73:1155-1164, 1993, the disclosure of which is incorporated herein byreference in its entirety). Briefly, a nucleotide sequence of interestto be inserted in a targeted location of the genome harbors at least twoloxP sites in the same orientation and located at the respective ends ofa nucleotide sequence to be excised from the recombinant genome. Theexcision event requires the presence of the recombinase (Cre) enzymewithin the nucleus of the recombinant cell host. The recombinase enzymemay be brought at the desired time either by (a) incubating therecombinant cell hosts in a culture medium containing this enzyme, byinjecting the Cre enzyme directly into the desired cell, such asdescribed by Araki et al. (Proc. Natl; Acad. Sci. USA, 92: 160-164,1995, the disclosure of which is incorporated herein by reference in itsentirety), or by lipofection of the enzyme into the cells, such asdescribed by Baubonis et al. (Nucleic Acids Res., 21:2025-2029, 1993,the disclosure of which is incorporated herein by reference in itsentirety); (b) transfecting the cell host with a vector comprising theCre coding sequence operably linked to a promoter functional in therecombinant cell host, which promoter being optionally inducible, saidvector being introduced in the recombinant cell host, such as describedby Gu et al. (Cell, 73:1155-1164, 1993, the disclosure of which isincorporated herein by reference in its entirety) and Sauer et al.(Proc. Natl; Acad. Sci. USA, 85:5166-5170, 1988, the disclosure of whichis incorporated herein by reference in its entirety); (c) introducing inthe genome of the cell host a polynucleotide comprising the Cre codingsequence operably linked to a promoter functional in the recombinantcell host, which promoter is optionally inducible, and saidpolynucleotide being inserted in the genome of the cell host either by arandom insertion event or an homologous recombination event, such asdescribed by Gu et al. (Science, 265:103-106, 1994, the disclosure ofwhich is incorporated herein by reference in its entirety).

[0229] In the specific embodiment wherein the vector containing thesequence to be inserted in the 12-LO gene by homologous recombination isconstructed in such a way that selectable markers are flanked by loxPsites of the same orientation, it is possible, by treatment by the Creenzyme, to eliminate the selectable markers while leaving the 12-LOsequences of interest that have been inserted by an homologousrecombination event. Again, two selectable markers are needed: apositive selection marker to select for the recombination event and anegative selection marker to select for the homologous recombinationevent. Vectors and methods using the Cre-loxP system are furtherdescribed by Zou et al. (Curr. Biol., 4:1099-1103, 1994), the disclosureof which is incorporated herein by reference in its entirety.

[0230] Thus, a third preferred DNA construct of the invention comprises,from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprisedin the 12-LO genomic sequence; (b) a nucleotide sequence comprising apolynucleotide encoding a positive selection marker, said nucleotidesequence comprising additionally two sequences defining a siterecognized by a recombinase, such as a loxP site, the two sites beingplaced in the same orientation; and (c) a second nucleotide sequencethat is comprised in the 12-LO genomic sequence, and is located on thegenome downstream of the first 12-LO nucleotide sequence (a).

[0231] The sequences defining a site recognized by a recombinase, suchas a loxP site, are preferably located within the nucleotide sequence(b) at suitable locations bordering the nucleotide sequence for whichthe conditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event.

[0232] In a preferred embodiment of a method using the third DNAconstruct described above, the excision of the polynucleotide fragmentbordered by the two sites recognized by a recombinase, preferably twoloxP sites, is performed at a desired time, due to the presence withinthe genome of the recombinant cell host of a sequence encoding the Creenzyme operably linked to a promoter sequence, preferably an induciblepromoter, more preferably a tissue-specific promoter sequence and mostpreferably a promoter sequence which is both inducible andtissue-specific, such as described by Gu et al. (Science, 265:103-106,1994), the disclosure of which is incorporated herein by reference inits entirety.

[0233] The presence of the Cre enzyme within the genome of therecombinant cell host may result of the breeding of two transgenicanimals, the first transgenic animal bearing the 12-LO-derived sequenceof interest containing the loxP sites as described above and the secondtransgenic animal bearing the Cre coding sequence operably linked to asuitable promoter sequence, such as described by Gu et al. (Science,265:103-106, 1994), the disclosure of which is incorporated herein byreference in its entirety.

[0234] Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton et al. (J.Virol., 69:4600-4606, 1995) and Kanegae et al. (Nucleic Acids Res.,23:3816-3821, 1995), the disclosures of which are incorporated herein byreference in their entireties.

[0235] The DNA constructs described above may be used to introduce adesired nucleotide sequence of the invention, preferably a 12-LO genomicsequence or a 12-LO cDNA sequence, and by most preferably an alteredcopy of a 12-LO genomic or cDNA sequence, within a predeterminedlocation of the targeted genome, leading either to the generation of analtered copy of a targeted gene (knock-out homologous recombination) orto the replacement of a copy of the targeted gene by another copysufficiently homologous to allow an homologous recombination event tooccur (knock-in homologous recombination).

[0236] Recombinant Vectors

[0237] The term “vector” is used herein to designate either a circularor a linear DNA or RNA molecule, which is either double-stranded orsingle-stranded, and which comprise at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

[0238] The present invention encompasses a family of recombinant vectorsthat comprise a regulatory polynucleotide derived from the 12-LO genomicsequence, or a coding polynucleotide from the 12-LO genomic sequence.Consequently, the present invention further deals with a recombinantvector comprising either a regulatory polynucleotide comprised in thenucleic acid of SEQ ID No. 651 or a polynucleotide comprising the 12-LOcoding sequence or both.

[0239] In a first preferred embodiment, a recombinant vector of theinvention is used to amplify the inserted polynucleotide derived from a12-LO genomic sequence selected from the group consisting of the nucleicacids of SEQ ID No. 651 or a 12-LO cDNA, for example the cDNA of SEQ IDNo. 652 in a suitable host cell, this polynucleotide being amplifiedeach time the recombinant vector replicates. Generally, a recombinantvector of the invention may comprise any of the polynucleotidesdescribed herein, including regulatory sequences and coding sequences,as well as any 12-LO primer or probe as defined above.

[0240] In a second preferred embodiment, recombinant vectors of theinvention consist of expression vectors comprising either a regulatorypolynucleotide or a coding nucleic acid of the invention, or both.Within certain embodiments, expression vectors are employed to expressthe 12-LO polypeptide which can be then purified and, for example beused in ligand screening assays or as an immunogen in order to raisespecific antibodies directed against the 12-LO protein. In otherembodiments, the expression vectors are used for constructing transgenicanimals and also for gene therapy. Expression requires that appropriatesignals are provided in the vectors, said signals including variousregulatory elements, such as enhancers/promoters from both viral andmammalian sources that drive expression of the genes of interest in hostcells. Dominant drug selection markers for establishing permanent,stable cell clones expressing the products are generally included in theexpression vectors of the invention, as they are elements that linkexpression of the drug selection markers to expression of thepolypeptide.

[0241] More particularly, the present invention relates to expressionvectors which include nucleic acids encoding a 12-LO protein, preferablythe 12-LO protein of the amino acid sequence of SEQ ID No. 653, underthe control of a regulatory sequence selected among the12-LO regulatorypolynucleotides of SEQ ID Nos. 651 and 652, or alternatively under thecontrol of an exogenous regulatory sequence.

[0242] Consequently, preferred expression vectors of the invention areselected from the group consisting of: (a) the 12-LO regulatory sequencecomprised therein drives the expression of a coding polynucleotideoperably linked thereto; (b) the 12-LO coding sequence is operablylinked to regulation sequences allowing its expression in a suitablecell host and/or host organism.

[0243] Additionally, the recombinant expression vector described abovemay also comprise a nucleic acid comprising a 5′-regulatorypolynucleotide, preferably a 5′-regulatory polynucleotide of the 12-LOgene. Additionally, the recombinant expression vector described abovemay also comprise a nucleic acid comprising a 3′-regulatorypolynucleotide, preferably a 3′-regulatory polynucleotide of the 12-LOgene. The 12-LO 3′-regulatory polynucleotide may also comprise the3′-UTR sequence contained in the nucleotide sequence of SEQ ID No. 652.The 5′-regulatory polynucleotide may also include the 5′-UTR sequence ofthe 12-LO cDNA, or a biologically active fragment or variant thereof.The invention also pertains to a recombinant expression vector usefulfor the expression of the 12-LO coding sequence, wherein said vectorcomprises a nucleic acid of SEQ ID No. 652.

[0244] The invention also relates to a recombinant expression vectorcomprising a nucleic acid comprising the nucleotide sequence beginningat the nucleotide in position 40 and ending in position 2028 of thepolynucleotide of SEQ ID No. 652.

[0245] Some of the elements which can be found in the vectors of thepresent invention are described in further detail in the followingsections.

[0246] 1. General Features of the Expression Vectors of the Invention.

[0247] A recombinant vector according to the invention comprises, but isnot limited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic and synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

[0248] (1) a genetic element or elements having a regulatory role ingene expression, for example promoters or enhancers. Enhancers arecis-acting elements of DNA, usually from about 10 to 300 bp in lengththat act on the promoter to increase the transcription.

[0249] (2) a structural or coding sequence which is transcribed intoMnRNA and eventually translated into a polypeptide, said structural orcoding sequence being operably linked to the regulatory elementsdescribed in (1); and

[0250] (3) appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems preferably include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, when a recombinant protein is expressed without a leaderor transport sequence, it may include a N-terminal residue. This residuemay or may not be subsequently cleaved from the expressed recombinantprotein to provide a final product.

[0251] Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsite, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

[0252] The in vivo expression of a 12-LO polypeptide of SEQ ID Nos. 653and 654 may be useful in order to correct a genetic defect related tothe expression of the native gene in a host organism or to theproduction of a biologically inactive 12-LO protein.

[0253] Consequently, the present invention also deals with recombinantexpression vectors mainly designed for the in vivo production of the12-LO polypeptide of SEQ I) Nos. 653-654 or fragments or variantsthereof by the introduction of the appropriate genetic material in theorganism of the patient to be treated. This genetic material may beintroduced in vitro in a cell that has been previously extracted fromthe organism, the modified cell being subsequently reintroduced in thesaid organism, directly in vivo into the appropriate tissue.

[0254] 2. Regulatory Elements.

[0255] The suitable promoter regions used in the expression vectorsaccording to the present invention are chosen taking into account thecell host in which the heterologous gene has to be expressed. Theparticular promoter employed to control the expression of a nucleic acidsequence of interest is not believed to be important, so long as it iscapable of directing the expression of the nucleic acid in the targetedcell. Thus, where a human cell is targeted, it is preferable to positionthe nucleic acid coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell, such as,for example, a human or a viral promoter.

[0256] A suitable promoter may be heterologous with respect to thenucleic acid for which it controls the expression or alternatively canbe endogenous to the native polynucleotide containing the codingsequence to be expressed. Additionally, the promoter is generallyheterologous with respect to the recombinant vector sequences withinwhich the construct promoter/coding sequence has been inserted.

[0257] Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors.

[0258] Preferred bacterial promoters are the LacI, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trppromoters (EP 0036776, the disclosure of which is incorporated herein byreference in its entirety), the polyhedrin promoter, or the p10 proteinpromoter from baculovirus (Kit Novagen) (Smith et al., Mol.Cell.Biol.3:2156-2165, 1983; O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual., W.H. Freeman andCo., New York, 1992, thedisclosures of which are incorporated herein by reference in theirentireties), the lambda PR promoter or also the trc promoter.

[0259] Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-L. Selection of a convenient vector and promoter is wellwithin the level of ordinary skill in the art.

[0260] The choice of a promoter is well within the ability of a personskilled in the field of genetic egineering. For example, one may referto the book of Sambrook et al. (Molecular Cloning: A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989), the disclosure of which is incorporated herein by reference inits entirety.

[0261] Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

[0262] The vector containing the appropriate DNA sequence as describedabove, more preferably 12-LO gene regulatory polynucleotide, apolynucleotide encoding the 12-LO polypeptide of SEQ ID Nos. 653 and 654or both of them, can be utilized to transform an appropriate host toallow the expression of the desired polypeptide or polynucleotide.

[0263] 3. Selectable Markers.

[0264] Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. The selectable marker genes for selection of transformed hostcells are preferably dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline,rifampicin or ampicillin resistance in E. coli, or levan saccharase formycobacteria, this latter marker being a negative selection marker.

[0265] 4. Preferred Vectors.

[0266] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and abacterial origin of replication derived from commercially availableplasmids comprising genetic elements of pBR322 (ATCC 37017). Suchcommercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala,Sweden), and GEM1 (Promega Biotec, Madison, Wis., USA). Large numbers ofother suitable vectors are known to those of skill in the art, andcommercially available, such as the following bacterial vectors : pQE70,pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK,pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTI, pSG(Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (Q1Aexpress).

[0267] The P1 bacteriophage vector may contain large inserts rangingfrom about 80 to about 100 kb. The construction of PI bacteriophagevectors such as p158 or p158/neo8 are described by Sternberg (Mamm.Genome, 5:397-404, 1994), the disclosure of which is incorporated hereinby reference in its entirety. Recombinant P1 clones comprising 12-LOnucleotide sequences may be designed for inserting large polynucleotidesof more than 40 kb (Linton etal., J. Clin. Invest., 92:3029-3037, 1993),the disclosure of which is incorporated herein by reference in itsentirety. To generate P1 DNA for transgenic experiments, a preferredprotocol is the protocol described by McCormick et al. (Genet. Anal.Tech. Appl., 11:158-164, 1994). Briefly, E. coli (preferably strainNS3529) harboring the P1 plasmid are grown overnight in a suitable brothmedium containing 25 μg/ml of kanamycin. The P1 DNA is prepared from theE. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen,Chatsworth, Calif., USA), according to the manufacturer's instructions.The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500columns, using the washing and elution buffers contained in the kit. Aphenol/chloroform extraction is then performed before precipitating theDNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl,pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed byspectrophotometry.

[0268] When the goal is to express a P1 clone comprising 12-LOnucleotide sequences in a transgenic animal, typically in transgenicmice, it is desirable to remove vector sequences from the P1 DNAfragment, for example by cleaving the P1 DNA at rare-cutting siteswithin the P1 polylinker (SfiI, NotI or SalI). The P1 insert is thenpurified from vector sequences on a pulsed-field agarose gel, usingmethods similar using methods similar to those originally reported forthe isolation of DNA from YACs (Schedl et al., Nature 362:258-261 1993;Peterson et al., Proc. Natl. Acad. Sci. USA 90:7593-7597, 1993, thedisclosures of which are incorporated herein by reference in theirentireties). At this stage, the resulting purified insert DNA can beconcentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit(Millipore, Bedford, Mass., USA—30,000 molecular weight limit) and thendialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μMEDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on amicrodyalisis membrane (type VS, 0.025 μM from Millipore). Theintactness of the purified P1 DNA insert is assessed by electrophoresison 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel andstaining with ethidium bromide.

[0269] A suitable vector for the expression of the 12-LO polypeptide ofSEQ ID Nos. 653 and 654 is a baculovirus vector that can be propagatedin insect cells and in insect cell lines. A specific suitable hostvector system is the pVL1392/1393 baculovirus transfer vector(Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL1711) which is derived from Spodoptera frugiperda.

[0270] Other suitable vectors for the expression of the 12-LOpolypeptide of SEQ ID Nos. 653 and 654 in a baculovirus expressionsystem include those described by Chai et al. (Biotech. Appl. Biochem.,18:259-273, 1993), Vlasak etal. (Eur. J. Biochem., 135: 123-126, 1983)and Lenhard et al. (Gene, 169: 187-190, 1996), the disclosures of whichare incorporated herein by reference in their entireties.

[0271] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery systems ofchoice for the transfer of exogenous polynucleotides in vivo,particularly to mammals, including humans. These vectors provideefficient delivery of genes into cells, and the transferred nucleicacids are stably integrated into the chromosomal DNA of the host.

[0272] Particularly preferred retroviruses for the preparation orconstruction of retroviral in vitro or in vitro gene delivery vehiclesof the present invention include retroviruses selected from the groupconsisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Gross(ATCC No.

[0273] VR-590), Rauscher (ATCC No. VR-998) and Moloney Murine LeukemiaVirus (ATCC No. VR-190; PCT Application No. WO 94/24298, the disclosureof which is incorporated herein by reference in its entirety).Particularly preferred Rous Sarcoma Viruses include Bryan high titer(ATCC Nos. VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferredretroviral vectors are those described in Roth et al. (Nature Medicine,2:985-991, 1996), PCT Application No. WO 93/25234 and PCT ApplicationNo. WO 94/ 06920, the disclosures of which are incorporated herein byreference in their entireties.

[0274] Yet another viral vector system that is contemplated by theinvention consists in the adeno-associated virus (AAV). Theadeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle(Muzyczka et al., Current Topics in Microbiol. Immunol., 158:97-129,1992, the disclosure of which is incorporated herein by reference in itsentirety). It is also one of the few viruses that may integrate its DNAinto non-dividing cells, and exhibits a high frequency of stableintegration (McLaughlin et al., Am. J. Hum. Genet., 59: 561-569, 1989,the disclosure of which is incorporated herein by reference in itsentirety). One advantageous feature of AAV derives from its reducedefficacy for transducing primary cells relative to transformed cells.

[0275] The bacterial artificial chromosome (BAC) cloning system (Shizuyaet al., Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797, 1992, the disclosureof which is incorporated herein by reference in its entirety) has beendeveloped to stably maintain large fragments of genomic DNA (100-300 kb)in E. coli. A preferred BAC vector consists of pBeloBAC 11 vector thathas been described by Kim et al. (Genomics, 34:213-218,1996), thedisclosure of which is incorporated herein by reference in its entirety.BAC libraries are prepared with this vector using size-selected genomicDNA that has been partially digested using enzymes that permit ligationinto either the Bam HI or HindIII sites in the vector. Flanking thesecloning sites are T7 and SP6 RNA polymerase transcription initiationsites that can be used to generate end probes by either RNAtranscription or PCR methods. After the construction of a BAC library inE. coli, BAC DNA is purified from the host cell as a supercoiled circle.Converting these circular molecules into a linear form precedes bothsize determination and introduction of the BACs into recipient cells.The cloning site is flanked by two Not I sites, permitting clonedsegments to be excised from the vector by Not I digestion.Alternatively, the DNA insert contained in the pBeloBAC II vector may belinearized by treatment of the BAC vector with the commerciallyavailable enzyme lambda terminase that leads to the cleavage at theunique cosN site, but this cleavage method results in a full length BACclone containing both the insert DNA and the BAC sequences.

[0276] 5. Delivery of the Recombinant Vectors.

[0277] In order to effect expression of the polynucleotides andpolynucleotide constructs of the invention, these constructs must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cell lines, or in vivo or exvivo, as in the treatment of certain diseases states. One mechanism isviral infection where the expression construct is encapsidated in aninfectious viral particle.

[0278] Several non-viral methods for the transfer of polynucleotidesinto cultured mammalian cells are also contemplated by the presentinvention, and include, without being limited to, calcium phosphateprecipitation (Chen et al., Proc. Natl. Acad. Sci. USA, 94:10756-10761,1987, the disclosure of which is incorporated herein by reference in itsentirety), DEAE-dextran (Gopal, Mol. Cell. Biol., 5:1188-1190, 1985, thedisclosure of which is incorporated herein by reference in itsentirety), electroporation (Tur-Kaspa et al., Mol. Cell. Biol.,6:716-718, 1986, the disclosure of which is incorporated herein byreference in its entirety), direct microinjection (Harland et al., J.Cell. Biol. 101:1094-1095, 1985), DNA-loaded liposomes (Nicolau et al.,Biochim. Biophys. Acta. 721:185-190,1982; Fraley et al., Natl. Acad.Sci. USA 76:3348-3352, 1979, the disclosures of which are incorporatedherein by reference in their entireties), and receptor-mediatetransfection (Wu and Wu, J. Biol. Chem. 262:44294432, 1987; Wu and WuBiochemistry 27:887-892, 1988, the disclosures of which are incorporatedherein by reference in their entireties). Some of these techniques maybe successfully adapted for in vivo or ex vivo use.

[0279] Once the expression polynucleotide has been delivered into thecell, it may be stably integrated into the genome of the recipient cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

[0280] One specific embodiment for a method for delivering a protein orpeptide to the interior of a cell of a vertebrate in vivo comprises thestep of introducing a preparation comprising a physiologicallyacceptable carrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

[0281] Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and in PCT application No. WO 95/11307, the disclosures of whichare incorporated herein by reference in their entireties.

[0282] In still another embodiment of the invention, the transfer of anaked polynucleotide of the invention, including a polynucleotideconstruct of the invention, into cells may be proceeded with a particlebombardment (biolistic), said particles being DNA-coatedmicroprojectiles accelerated to a high velocity allowing them to piercecell membranes and enter cells without killing them, such as describedby Klein et al. (Nature 327:70-73, 1987), the disclosure of which isincorporated herein by reference in its entirety.

[0283] In a further embodiment, the polynucleotide of the invention maybe entrapped in a liposome (Ghosh and Bacchawat, Targeting of liposomesto hepatocytes, In: Liver Diseases, Targeted diagnosis and therapy usingspecific rceptors and ligands, Marcel Dekeker, New York, 87-104, 1991;Wong et al., Gene 10:87-94, 1980; Nicolau et al., Biochim. Biophys.Acta. 721:185-190, 1982, the disclosures of which are incorporatedherein by reference in their entireties)

[0284] In a specific embodiment, the invention provides a compositionfor the in vivo production of the 12-LO protein or polypeptide describedherein. It comprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said protein or polypeptide.

[0285] The amount of vector to be injected to the desired host organismvaries according to the site of injection. As an indicative dose, itwill be injected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

[0286] In another embodiment of the vector according to the invention,it may be introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired 12-LOpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

[0287] Host Cells

[0288] Another object of the invention consists of a host cell that havebeen transformed or transfected with one of the polynucleotidesdescribed therein, and more precisely a polynucleotide either comprisinga 12-LO regulatory polynucleotide or the coding sequence of the 12-LOpolypeptide having the amino acid sequence of SEQ ID Nos. 653 or 654.Are included host cells that are transformed (prokaryotic cells) or thatare transfected (eukaryotic cells) with a recombinant vector such as oneof those described above.

[0289] Generally, a recombinant host cell of the invention comprises anyone of the polynucleotides or the recombinant vectors described therein.

[0290] A preferred recombinant host cell according to the inventioncomprises a polynucleotide selected from the following group ofpolynucleotides:

[0291] a) a purified or isolated nucleic acid encoding a 12-LOpolypeptide, or a polypeptide fragment or variant thereof.

[0292] b) a purified or isolated nucleic comprising at least 8,preferably at least 15, more preferably at least 25, consecutivenucleotides of the nucleotide sequence SEQ ID No. 651, a nucleotidesequence complementary thereto, or a variant thereof.

[0293] c) a purified or isolated nucleic acid comprising at least 8consecutive nucleotides, preferably at least 15, more preferably atleast 25 of the nucleotide sequence SEQ ID No. 652, a nucleotidesequence complementary thereto or a variant thereof.

[0294] d) a purified or isolated nucleic acid comprising an exon of the12-LO gene, a sequence complementary thereto or a fragment or a variantthereof.

[0295] e) a purified or isolated nucleic acid comprising a combinationof at least two exons of the12-LO gene, or the sequences complementarythereto wherein the polynucleotides are arranged within the nucleicacid, from the 5′ end to the 3′end of said nucleic acid, in the sameorder than in SEQ ID No. 651.

[0296] f) a purified or isolated nucleic acid comprising the nucleotidesequence SEQ ID No. 651 or the sequences complementary thereto or abiologically active fragment thereof.

[0297] g) a polynucleotide consisting of:

[0298] (1) a nucleic acid comprising a regulatory polynucleotide of SEQID No. 651 or the sequences complementary thereto or a biologicallyactive fragment thereof

[0299] (2) a polynucleotide encoding a desired polypeptide or nucleicacid.

[0300] i) a DNA construct as described previously in the presentspecification.

[0301] Another preferred recombinant cell host according to the presentinvention is characterized in that its genome or genetic background(including chromosome, plasmids) is modified by the nucleic acid codingfor the 12-LO polypeptide of SEQ ID Nos. 653 and 654 or fragments orvariants thereof.

[0302] Preferred host cells used as recipients for the expressionvectors of the invention are the following:

[0303] a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-αstrain), Bacillus subtilis, Salmonella typhimurium, and strains fromspecies like Pseudomonas, Streptomyces and Staphylococcus..

[0304] b) Eukaryotic host cells: HeLa cells (ATCC N°CCL2; N°CCL2.1;N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650;N°CRL1651), Sf-9 cells (ATCC N°CRL1711), C127 cells (ATCC N°CRL-1804),3T3 (ATCC N°CRL-6361), CHO (ATCC N°CCL-61), human kidney 293.(ATCCN°45504; N°CRL-1573) and BHK (ECACC N°84100501; N°84111301)

[0305] c) Other mammalian host cells:

[0306] The 12-LO gene expression in mammalian, and typically human,cells may be rendered defective, or alternatively it may be proceededwith the insertion of a 12-LO genomic or cDNA sequence with thereplacement of the 12-LO gene counterpart in the genome of an animalcell by a 12-LO polynucleotide according to the invention. These geneticalterations may be generated by homologous recombination events usingspecific DNA constructs that have been previously described.

[0307] One kind of host cell that may be used is mammalian zygotes, suchas murine zygotes. For example, murine zygotes may undergomicroinjection with a purified DNA molecule of interest, such as apurified DNA molecule that has previously been adjusted to aconcentration range from 1 ng/ml (for BAC inserts) 3 ng/μl (for P1bacteriophage inserts) in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing100 mM NaCl, 30 μM spermine, and70 μM spermidine. When the DNA to bemicroinjected is relatively large, polyamines and high saltconcentrations can be used to avoid mechanical breakage of this DNA, asdescribed by Schedl et al. (Nucleic Acids Res. 21:4783-4787, 1993), thedisclosure of which is incorporated herein by reference in its entirety.

[0308] Anyone of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC n°CRL-1821), ES-D3 (ATCC n° CRL1934 and n° CRL-1 1632),YS001 (ATCC n° CRL-1 1776), 36.5 (ATCC n° CRL-11116). To maintain EScells in an uncommitted state, they are cultured in the presence ofgrowth inhibited feeder cells, which provide the appropriate signals topreserve this embryonic phenotype and serve as a matrix for ES celladherence. Preferred feeder cells consist of primary embryonicfibroblasts that are established from tissue of day 13- day 14 embryosof virtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (Methods in Enzymology, Academic Press,NewYork, 803-823, 1993), the disclosure of which is incorporated hereinby reference in its entirety, and are inhibited in growth byirradiation, such as described by Robertson (“Embryo-Derived StemCellLines,” E.J Robertson Ed.. Teratocarcinomas and Embrionic Stem Cells: APractical Approach. IRL Press, Oxford, 71, 1987), the disclosure ofwhich is incorporated herein by reference in its entirety, or by thepresence of an inhibitory concentration of LIF, such as described byPease and Williams (Exp. Cell. Res. 190:09-211, 1990), the disclosure ofwhich is incorporated herein by reference in its entirety.

[0309] The constructs in the host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.

[0310] Following transformation of a suitable host and growth of thehost to an appropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

[0311] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0312] Microbial cells employed in the expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known by the skill artisan.

[0313] Transgenic Animals

[0314] The terms “transgenic animals” or “host animals” used hereindesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. Preferred animals are non-human mammals and include thosebelonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats)and Oryctogalus (e.g. rabbits) which have their genome artificially andgenetically altered by the insertion of a nucleic acid according to theinvention.

[0315] The transgenic animals of the invention all include within aplurality of their cells a cloned recombinant or synthetic DNA sequence,more specifically one of the purified or isolated nucleic acidscomprising a 12-LO coding sequence, a 12-LO regulatory polynucleotide ora DNA sequence encoding an antisense polynucleotide such as described inthe present specification.

[0316] Preferred transgenic animals according to the invention containin their somatic cells and/or in their germ line cells a polynucleotideselected from the following group of polynucleotides:

[0317] a) a purified or isolated nucleic acid encoding a 12-LOpolypeptide, or a polypeptide fragment or variant thereof.

[0318] b) a purified or isolated nucleic comprising at least 8,preferably at least 15, more preferably at least 25, consecutivenucleotides of the nucleotide sequence SEQ ID No. 651, a nucleotidesequence complementary thereto.

[0319] c) a purified or isolated nucleic acid comprising at least 8consecutive nucleotides, preferably at least 15, more preferably atleast 25 of the nucleotide sequence SEQ ID No. 652, a nucleotidesequence complementary thereto.

[0320] d) a purified or isolated nucleic acid comprising an exon of the12-LO gene, a sequence complementary thereto or a fragment or a variantthereof.

[0321] e) a purified or isolated nucleic acid comprising a combinationof at least two exons of the 12-LO gene, or the sequences complementarythereto wherein the polynucleotides are arranged within the nucleicacid, from the 5′ end to the 3′end of said nucleic acid, in the sameorder than in SEQ ID No. 651.

[0322] f) a purified or isolated nucleic acid comprising the nucleotidesequence SEQ ID No. 651 or the sequences complementary thereto or abiologically active fragment thereof.

[0323] g) a polynucleotide consisting of:

[0324] (1) a nucleic acid comprising a regulatory polynucleotide of SEQID No. 651 or the sequences complementary thereto or a biologicallyactive fragment thereof

[0325] (2) a polynucleotide encoding a desired polypeptide or nucleicacid.

[0326] i) a DNA construct as described previously in the presentspecification.

[0327] The transgenic animals of the invention thus contain specificsequences of exogenous genetic material such as the nucleotide sequencesdescribed above in detail.

[0328] In a first preferred embodiment, these transgenic animals may begood experimental models in order to study the diverse pathologiesrelated to cell differentiation, in particular concerning the transgenicanimals within the genome of which has been inserted one or severalcopies of a polynucleotide encoding a native 12-LO protein, oralternatively a mutant 12-LO protein.

[0329] In a second preferred embodiment, these transgenic animals mayexpress a desired polypeptide of interest under the control of theregulatory polynucleotides of the 12-LO gene, leading to good yields inthe synthesis of this protein of interest, and eventually a tissuespecific expression of this protein of interest.

[0330] The design of the transgenic animals of the invention may be madeaccording to the conventional techniques well known for one skilled inthe art. For more details regarding the production of transgenicanimals, and specifically transgenic mice, one may refer to U.S. Pat.Nos. 4,873,191, issued Oct.10, 1989, 5,464,764 issued Nov. 7, 1995 and5,789,215, issued Aug. 4, 1998, these documents being hereinincorporated by reference in their entireties to disclose methodsproducing transgenic mice.

[0331] Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thathas incorporated exogenous genetic material. The procedure involvesobtaining the genetic material, or a portion thereof, which encodeseither a 12-LO coding sequence, a 12-LO regulatory polynucleotide or aDNA sequence encoding a 12-LO antisense polynucleotide such as describedin the present specification.

[0332] A recombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. The insertion is preferably made usingelectroporation, such as described by Thomas et al. (Cell 51:503-512,1987), the disclosure of which is incorporated herein by reference inits entirety. The cells subjected to electroporation are screened (e.g.by selection via selectable markers, by PCR or by Southern blotanalysis) to find positive cells -which have integrated the exogenousrecombinant polynucleotide into their genome, preferably via anhomologous recombination event. An illustrative positive-negativeselection procedure that may be used according to the invention isdescribed by Mansour et al. (Nature 336:348-352, 1988), the disclosureof which is incorporated herein by reference in its entirety.

[0333] Then, the positive cells are isolated, cloned and injected into3.5 days old blastocysts from mice, such as described by Bradley(“Production and Analysis of Chimaeric Mice,” E. J Robertson (Ed.),Teratocarcinomas and embryonic stem cells: A practical approach IRLPress, Oxford, 113, 1987), the disclosure of which is incorporatedherein by reference in its entirety. The blastocysts are then insertedinto a female host animal and allowed to grow to term.

[0334] Alternatively, the positive ES cells are brought into contactwith embryos at the 2.5 days old 8-16 cell stage (morulae) such asdescribed by Wood et al. (Proc. Natl. Acad. Sci. U.S.A. 90:4582-4585,1993) or by Nagy et al. (Proc. Natl. Acad. Sci. USA. 90: 8424-8428,1993), the disclosures of which are incorporated herein by reference intheir entireties, the ES cells being internalized to colonizeextensively the blastocyst including the cells which will give rise tothe germ line.

[0335] The offspring of the female host are tested to determine whichanimals are transgenic e.g. include the inserted exogenous DNA sequenceand which are wild-type. Thus, the present invention also concerns atransgenic animal containing a nucleic acid, a recombinant expressionvector or a recombinant host cell according to the invention.

[0336] A further object of the invention consists of recombinant hostcells obtained from a transgenic animal described herein.

[0337] Recombinant cell lines may be established in vitro from cellsobtained from any tissue of a transgenic animal according to theinvention, for example by transfection of primary cell cultures withvectors expressing onc-genes such as SV40 large T antigen, as describedby Chou (Mol. Endocrinol. 3:1511-1514, 1989) and Shay et al. (Biochem.Biophys. Acta. 1072:1-7, 1991), the disclosures of which areincorporated herein by reference in their entireties.

[0338] E. 12-Lipoxygenase Polypeptides

[0339] The term “12-LO polypeptides” is used herein to embrace all ofthe proteins and polypeptides of the present invention. Also formingpart of the invention are polypeptides encoded by the polynucleotides ofthe invention, as well as fusion polypeptides comprising suchpolypeptides. The invention embodies 12-LO proteins from humans,including isolated or purified 12-LO proteins consisting, consistingessentially, or comprising the sequence of SEQ ID Nos. 653 and 654.

[0340] Biallelic markers are associated with amino acid substitutions inthe polypeptide sequence of 12-LO. It should be noted the 12-LO proteinsof the invention are based on the naturally-occurring variants of theamino acid sequence of human 12-LO; wherein the Arg residue of aminoacid position 189 has been replaced with a His residue (biallelic marker10-346-141), the Asp residue of amino acid position 225 has beenreplaced with a His residue (biallelic marker 10-347-111), the Argresidue of amino acid position 243 has been replaced with a Cys residue(biallelic marker 10-347-165), the Gln residue of amino acid position261 has been replaced with an Arg residue (biallelic marker 10-347-220),the Ser residue of amino acid position 322 has been replaced with a Asnresidue (biallelic marker 10-349-97), the Pro residue of amino acidposition 337 has been replaced with an Arg residue (biallelic marker10-349-142), the Thr residue of amino acid position 568 has beenreplaced with an Asn residue (biallelic marker 10-340-112) and whereinthe Met residue of amino acid position 574 has been replaced with a Lysresidue (biallelic marker 10-340-112). Variant proteins and thefragments thereof which contain amino acid position 189 are collectivelyreferred to herein as “189-His variants.” Variant proteins and thefragments thereof which contain amino acid position 225 are collectivelyreferred to herein as “225-His variants.” Variant proteins and thefragments thereof which, contain amino acid position 243, arecollectively referred to herein as “243-Cys variants.” Variant proteinsand the fragments thereof which contain amino acid position 261 arecollectively referred to herein as “261-Arg variants.” Variant proteinsand the fragments thereof which contain amino acid position 322 arecollectively referred to herein as “322-Asn variants.” Variant proteinsand the fragments thereof which contain amino acid position 337 arecollectively referred to herein as “337-Arg variants.” Variant proteinsand the fragments thereof which contain amino acid position 568 arecollectively referred to herein as “568-Asn variants.” Variant proteinsand the fragments thereof which contain amino acid position 574 arecollectively referred to herein as “574-Lys variants.” In each of theseamino acid substitutions the original residue is replaced by anon-equivalent amino acid presenting different chemical properties.Therefore, these substitutions cause alterations in the activity,specificity and function of the 12-LO enzyme.

[0341] One allele of biallelic marker 10-349-216 is associated with thedeletion of a Leu residue at amino acid position 362 of SEQ ID No. 653.12-LO polypeptides of the present invention also include 12-LOpolypeptides wherein the Leu residue at amino acid position 362 of SEQID No. 653 has been deleted.

[0342] One allele of biallelic marker 10-343-231 is associated with aframeshift in the open reading frame of the 12-LO gene leading to theexpression of the variant 12-LO polypeptide of SEQ ID No. 654.

[0343] The present invention embodies isolated, purified, andrecombinant polypeptides comprising a contiguous span of at least 6amino acids, preferably at least 8 to 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653,wherein said contiguous span comprises at least one amino acid positionselected from the group consisting of: an His residue et amino acidposition 189, an His residue at amino acid position 225, a Cys residueat amino acid position 243, an Arg residue at amino acid position 261,an Asn residue at amino acid position 322, an Arg residue at amino acidposition 337, a Asn residue at amino acid position 362, an Asn at aminoacid position 568 and a Lys residue at amino acid position 574.

[0344] The present invention further provides isolated, purified, andrecombinant polypeptides comprising a contiguous span of at least 6amino acids, preferably at least 8 to 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 654,wherein said contiguous span comprises at least one of amino acidpositions 110-131 of SEQ ID No. 654.

[0345] The present invention further embodies isolated, purified, andrecombinant polypeptides comprising a contiguous span of at least 6amino acids, preferably at least 8 to 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653,wherein said contiguous span comprises a Leu residue at amino acidposition 389 of SEQ ID No. 653.

[0346] In other preferred embodiments the contiguous stretch of aminoacids comprises the site of a mutation or functional mutation, includinga deletion, addition, swap or truncation of the amino acids in the 12-LOprotein sequence.

[0347] 12-LO proteins are preferably isolated from human or mammaliantissue samples or expressed from human or mammalian genes. The 12-LOpolypeptides of the invention can be made using routine expressionmethods known in the art. The polynucleotide encoding the desiredpolypeptide is ligated into an expression vector suitable for anyconvenient host. Both eukaryotic and prokaryotic host systems are usedin forming recombinant polypeptides. The polypeptide is then isolatedfrom lysed cells or from the culture medium and purified to the extentneeded for its intended use. Purification is by any technique known inthe art, for example, differential extraction, salt fractionation,chromatography, centrifugation, and the like. See, for example, Methodsin Enzymology for a variety of methods for purifying proteins.

[0348] In addition, shorter protein fragments are produced by chemicalsynthesis. Alternatively the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

[0349] Any 12-LO cDNA, including SEQ ID No. 652, is used to express12-LO proteins and polypeptides. The nucleic acid encoding the 12-LOprotein or polypeptide to be expressed is operably linked to a promoterin an expression vector using conventional cloning technology. The 12-LOinsert in the expression vector may comprise the full coding sequencefor the 12-LO protein or a portion thereof.

[0350] The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art. Commercially availablevectors and expression systems are available from a variety of suppliersincluding Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla,Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). Ifdesired, to enhance expression and facilitate proper protein folding,the codon context and codon pairing of the sequence is optimized for theparticular expression organism in which the expression vector isintroduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767,the disclosure of which is incorporated herein by reference in itsentirety.

[0351] In one embodiment, the entire coding sequence of the 12-LO cDNAthrough the poly A signal of the cDNA is operably linked to a promoterin the expression vector. Alternatively, if the nucleic acid encoding aportion of the 12-LO protein lacks a methionine to serve as theinitiation site, an initiating methionine can be introduced next to thefirst codon of the nucleic acid using conventional techniques.Similarly, if the insert from the 12-LO cDNA lacks a poly A signal, thissequence can be added to the construct by, for example, splicing out thePoly A signal from pSG5 (Stratagene) using Bg1I and Sa1I restrictionendonuclease enzymes and incorporating it into the mammalian expressionvector pXT1 (Stratagene). pXT1 contains the LTRs and a portion of thegag gene from Moloney Murine Leukemia Virus. The position of the LTRs inthe construct allow efficient stable transfection. The vector includesthe Herpes Simplex Thymidine Kinase promoter and the selectable neomycingene. The nucleic acid encoding the 12-LO protein or a portion thereofis obtained by PCR from a bacterial vector containing the 12-LO cDNA ofSEQ ID No. 652 using oligonucleotide primers complementary to the 12-LOcDNA or portion thereof and containing restriction endonucleasesequences for Pst I incorporated into the 5′primer and Bg1II at the 5′end of the corresponding cDNA 3′ primer, taking care to ensure that thesequence encoding the 12-LO protein or a portion thereof is positionedproperly with respect to the poly A signal. The purified fragmentobtained from the resulting PCR reaction is digested with PstI, bluntended with an exonuclease, digested with Bg1 II, purified and ligated topXT1, now containing a poly A signal and digested with Bg1II.

[0352] The ligated product is transfected into mouse NIH 3T3 cells usingLipofectin (Life Technologies, Inc., Grand Island, N.Y.) underconditions outlined in the product specification. Positive transfectantsare selected after growing the transfected cells in 600ug/ml G418(Sigma, St. Louis, Mo.).

[0353] Alternatively, the nucleic acids encoding the 12-LO protein or aportion thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge,Mass.). The resulting pED6dpc2 constructs is transfected into a suitablehost cell, such as COS 1 cells. Methotrexate resistant cells areselected and expanded.

[0354] The above procedures may also be used to express a mutant 12-LOprotein responsible for a detectable phenotype or a portion thereof.

[0355] The expressed proteins are purified using conventionalpurification techniques such as anmmonium sulfate precipitation orchromatographic separation based on size or charge. The protein encodedby the nucleic acid insert may also be purified using standardimmunochromatography techniques. In such procedures, a solutioncontaining the expressed 12-LO protein or portion thereof, such as acell extract, is applied to a column having antibodies against the 12-LOprotein or portion thereof is attached to the chromatography matrix. Theexpressed protein is allowed to bind the immunochromatography column.Thereafter, the column is washed to remove non-specifically boundproteins. The specifically bound expressed protein is then released fromthe column and recovered using standard techniques.

[0356] To confirm expression of the 12-LO protein or a portion thereof,the proteins expressed from host cells containing an expression vectorcontaining an insert encoding the 12-LO protein or a portion thereof canbe compared to the proteins expressed in host cells containing theexpression vector without an insert. The presence of a band in samplesfrom cells containing the expression vector with an insert which isabsent in samples from cells containing the expression vector without aninsert indicates that the 12-LO protein or a portion thereof is beingexpressed. Generally, the band will have the mobility expected for the12-LO protein or portion thereof. However, the band may have a mobilitydifferent than that expected as a result of modifications such asglycosylation, ubiquitination, or enzymatic cleavage.

[0357] Antibodies capable of specifically recognizing the expressed12-LO protein or a portion thereof, are described below.

[0358] If antibody production is not possible, the nucleic acidsencoding the 12-LO protein or a portion thereof is incorporated intoexpression vectors designed for use in purification schemes employingchimeric polypeptides. In such strategies the nucleic acid encoding the12-LO protein or a portion thereof is inserted in frame with the geneencoding the other half of the chimera. The other half of the chimera isβ-globin or a nickel binding polypeptide encoding sequence. Achromatography matrix having antibody to β-globin or nickel attachedthereto is then used to purify the chimeric protein. Protease cleavagesites is engineered between the β-globin gene or the nickel bindingpolypeptide and the 12-LO protein or portion thereof. Thus, the twopolypeptides of the chimera are separated from one another by proteasedigestion.

[0359] One useful expression vector for generating β-globin chimerics ispSG5 (Stratagene), which encodes rabbit β-globin. Intron II of therabbit β-globin gene facilitates splicing of the expressed transcript,and the polyadenylation signal incorporated into the construct increasesthe level of expression. These techniques are well known to thoseskilled in the art of molecular biology. Standard methods are publishedin methods texts such as Davis et al., (Basic Methods in MolecularBiology, L. G. Davis, M. D. Dibner, and J. F. Battey, ed., ElsevierPress, NY, 1986, the disclosure of which is incorporated herein byreference in its entirety) and many of the methods are available fromStratagene, Life Technologies, Inc., or Promega. Polypeptide mayadditionally be produced from the construct using in vitro translationsystems such as the In vitro Express™ Translation Kit (Stratagene).

[0360] F. Production Of Antibodies Against 12-Lipoxygenase Polypeptides

[0361] Any 12-LO polypeptide or whole protein may be used to generateantibodies capable of specifically binding to expressed 12-LO protein orfragments thereof as described. The antibody compositions of theinvention are capable of specifically binding to the 189-His variant ofthe 12-LO protein or, to the 225-His variant of the 12-LO protein or, tothe 243-Cys variant of the 12-LO protein or, to the 261-Arg variant ofthe 12-LO protein or, to the 322-Asn variant of the 12-LO or, to the337-Arg variant of the 12-LO protein or to the 574-Lys variant of the12-LO protein. A preferred embodiment of the invention encompassesisolated or purified antibody compositions capable of selectivelybinding, or which are capable of binding to an epitope-containingfragment of a polypeptide of the invention, wherein said epitopecomprises at least one amino acid position selected from the groupconsisting of an His residue et amino acid position 189, an His residueat amino acid position 225, a Cys residue at amino acid position 243, anArg residue at amino acid position 261, an Asn residue at amino acidposition 322, an Arg residue at amino acid position 337, a Asn residueat amino acid position 362, an Asn at amino acid position 568 and a Lysresidue at amino acid position 574. For an antibody composition tospecifically bind to these 12-LO variants it must demonstrate at least a5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for fulllength 189-His, 225-His, 243-Cys, 261-Arg, 322- Asn, 337-Arg or 574-Lysvariants in an ELISA, RIA, or other antibody-based binding assay than tofull length 12-LO proteins which have the alternative amino acidspecified in SEQ ID No. 653. Affinity of the antibody composition forthe epitope can further be determined by preparing competitive bindingcurves, as described, for example, by Fisher, D., (Manual of ClinicalImmunology, 2nd Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol.,Washington, D.C., Ch. 42, 1980), the disclosure of which is incorporatedherein by reference in its entirety.

[0362] Other preferred antibody compositions of the invention arecapable of specifically binding to amino acid positions 110-131 of SEQID No. 654.

[0363] The present invention also contemplates the use of polypeptidescomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 50,or 100 amino acids of a 12-LO polypeptide of SEQ ID No. 653 in themanufacture of antibodies, wherein said contiguous span comprises atleast one amino acid position selected from the group consisting of: anHis residue et amino acid position 189, an His residue at amino acidposition 225, a Cys residue at amino acid position 243, an Arg residueat amino acid position 261, an Asn residue at amino acid position 322,an Arg residue at amino acid position 337, a Asn residue at amino acidposition 362, an Asn at amino acid position 568 and a Lys residue atamino acid position 574.

[0364] In a preferred embodiment such polypeptides are useful in themanufacture of antibodies to detect the presence and absence of the189-His, 225-His, 243-Cys, 261-Arg, 322- Asn, 337-Arg, 568-Asn, or574-Lys variant.

[0365] The present invention further encompasses the use of isolated,purified, and recombinant polypeptides comprising a contiguous span ofat least 6 amino acids, preferably at least 8 to 10 amino acids, morepreferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids ofSEQ ID No. 654, wherein said contiguous span comprises at least one ofamino acid positions 110-131 of SEQ 25 ID No. 654.

[0366] In a preferred embodiment such polypeptides are useful in themanufacture of antibodies to detect the presence and absence of aminoacid positions 110-131 of SEQ ID No. 654.

[0367] Non-human animals or mammals, whether wild-type or transgenic,which express a different species of 12-LO than the one to whichantibody binding is desired, and animals which do not express 12-LO(i.e. an 12-LO knock out animal as described in herein) are particularlyuseful for preparing antibodies. 12-LO knock out animals will recognizeall or most of the exposed regions of 12-LO as foreign antigens, andtherefore produce antibodies with a wider array of 12-LO epitopes.Moreover, smaller polypeptides with only 10 to 30 amino acids may beuseful in obtaining specific binding to the 189-His, 225-His, 243-Cys,261-Arg, 322-Asn, 337-Arg, 568-Asn, or 574-Lys variants. In addition,the humoral immune system of animals which produce a species of 12-LOthat resembles the antigenic sequence will preferentially recognize thedifferences between the animal's native 12-LO species and the antigensequence, and produce antibodies to these unique sites in the antigensequence. Such a technique will be particularly useful in obtainingantibodies that specifically bind to the 189-His, 225-His, 243-Cys,261-Arg, 322- Asn, 337-Arg, 568-Asn, or 574-Lys variants. Thepreparation of antibody compositions is further described in Example 6.

[0368] Antibody preparations prepared according to the present inventionare useful in quantitative immunoassays which determine concentrationsof antigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body. The antibodies of the invention maybe labeled, either by a radioactive, a fluorescent or an enzymaticlabel. Consequently, the invention is also directed to a method fordetecting specifically the presence of a variant 12-LO polypeptideaccording to the invention in a biological sample, said methodcomprising the following steps : a) bringing into contact the biologicalsample with a polyclonal or monoclonal antibody that specifically bindsa variant 12-LO polypeptide or to a peptide fragment or variant thereof,and b) detecting the antigen-antibody complex formed. The invention alsoconcerns a diagnostic kit for detecting in vitro the presence of avariant 12-LO polypeptide according to the present invention in abiological sample, wherein said kit comprises:

[0369] a) a polyclonal or monoclonal antibody that specifically binds avariant 12-LO polypeptide or to a peptide fragment or variant thereof,optionally labeled;

[0370] b) a reagent allowing the detection of the antigen-antibodycomplexes formed, said reagent carrying optionally a label, or beingable to be recognized itself by a labeled reagent, more particularly inthe case when the above-mentioned monoclonal or polyclonal antibody isnot labeled by itself.

[0371] II. Methods for De Novo Identification of Biallelic Markers

[0372] Large fragments of human DNA, carrying genes of interest involvedin arachidonic acid metabolism; were cloned, sequenced and screened forbiallelic markers. Biallelic markers within the candidate genesthemselves as well as markers located on the same genomic fragment wereidentified. It will be clear to one of skill in the art that largefragments of human genomic DNA may be obtained from any appropriatesource and may be cloned into a number of suitable vectors.

[0373] In a preferred embodiment of the invention, BAC (BacterialArtificial Chromosomes) vectors were used to construct DNA librariescovering the entire human genome. Specific amplification primers weredesigned for each candidate gene and the BAC library was screened by PCRuntil there was at least one positive BAC clone per candidate gene.Genomic sequence, screened for biallelic markers, was generated bysequencing ends of BAC subclones. Details of a preferred embodiment areprovided in Example 1. As a preferred alternative to sequencing the endsof an adequate number of BAC subclones, high throughput deletion-basedsequencing vectors, which allow the generation of a high qualitysequence information covering fragments of about 6 kb, may be used.Having sequence fragments longer than 2.5 or 3 kb enhances the chancesof identifying biallelic markers therein. Methods of constructing andsequencing a nested set of deletions are disclosed in the related U.S.patent application entitled “High Throughput DNA Sequencing Vector”(Ser. No. 09/058,746).

[0374] In another embodiment of the invention, genomic sequences ofcandidate genes were available in public databases allowing directscreening for biallelic markers. Any of a variety of methods can be usedto screen a genomic fragment for single nucleotide polymorphisms such asdifferential hybridization with oligonucleotide probes, detection ofchanges in the mobility measured by gel electrophoresis or directsequencing of the amplified nucleic acid. A preferred method foridentifying biallelic markers involves comparative sequencing of genomicDNA fragments from an appropriate number of unrelated individuals.

[0375] In a first embodiment, DNA samples from unrelated individuals arepooled together, following which the genomic DNA of interest isamplified and sequenced. The nucleotide sequences thus obtained are thenanalyzed to identify significant polymorphisms. One of the majoradvantages of this method resides in the fact that the pooling of theDNA samples substantially reduces the number of DNA amplificationreactions and sequencing reactions, which must be carried out. Moreover,this method is sufficiently sensitive so that a biallelic markerobtained thereby usually demonstrates a sufficient frequency of its lesscommon allele to be useful in conducting association studies. Usually,the frequency of the least common allele of a biallelic markeridentified by this method is at least 10%.

[0376] In a second embodiment, the DNA samples are not pooled and aretherefore amplified and sequenced individually. This method is usuallypreferred when biallelic markers need to be identified in order toperform association studies within candidate genes. Preferably, highlyrelevant gene regions such as promoter regions or exon regions may bescreened for biallelic markers. A biallelic marker obtained using thismethod may show a lower degree of informativeness for conductingassociation studies, e.g. if the frequency of its less frequent allelemay be less than about 10%. Such a biallelic marker will however besufficiently informative to conduct association studies and it willfurther be appreciated that including less informative biallelic markersin the genetic analysis studies of the present invention, may allow insome cases the direct identification of causal mutations, which may,depending on their penetrance, be rare mutations.

[0377] The following is a description of the various parameters of apreferred method used by the inventors for the identification of thebiallelic markers of the present invention.

[0378] A. Genomic DNA Samples

[0379] The genomic DNA samples from which the biallelic markers of thepresent invention are generated are preferably obtained from unrelatedindividuals corresponding to a heterogeneous population of known ethnicbackground. The number of individuals from whom DNA samples are obtainedcan vary substantially, preferably from about 10 to about 1000, morepreferably from about 50 to about 200 individuals. Usually, DNA samplesare collected from at least about 100 individuals in order to havesufficient polymorphic diversity in a given population to identify asmany markers as possible and to generate statistically significantresults.

[0380] As for the source of the genomic DNA to be subjected to analysis,any test sample can be foreseen without any particular limitation. Thesetest samples include biological samples, which can be tested by themethods of the present invention described herein, and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcel specimens. The preferred source of genomic DNA used in the presentinvention is from peripheral venous blood of each donor. Techniques toprepare genomic DNA from biological samples are well known to theskilled technician. Details of a preferred embodiment are provided inExample 1. A person skilled in the art can choose to amplify pooled orunpooled DNA samples.

[0381] B. DNA Amplification

[0382] The identification of biallelic markers in a sample of genomicDNA may be facilitated through the use of DNA amplification methods. DNAsamples can be pooled or unpooled for the amplification step. DNAamplification techniques are well known to those skilled in the art.Various methods to amplify DNA fragments carrying biallelic markers arefurther described hereinafter in III.B. The PCR technology is thepreferred amplification technique used to identify new biallelicmarkers.

[0383] In a first embodiment, biallelic markers are identified usinggenomic sequence information generated by the inventors. Genomic DNAfragments, such as the inserts of the BAC clones described above, aresequenced and used to design primers for the amplification of 500 bpfragments. These 500 bp fragments are amplified from genomic DNA and arescanned for biallelic markers. Primers may be designed using the OSPsoftware (Hillier L. and Green P., Methods Appl. 1: 124-8, 1991). Allprimers may contain, upstream of the specific target bases, a commonoligonucleotide tail that serves as a sequencing primer. Those skilledin the art are familiar with primer extensions, which can be used forthese purposes.

[0384] In another embodiment of the invention, genomic sequences ofcandidate genes are available in public databases allowing directscreening for biallelic markers. Preferred primers, useful for theamplification of genomic sequences encoding the candidate genes, focuson promoters, exons and splice sites of the genes. A biallelic markerpresent in these functional regions of the gene has a higher probabilityto be a causal mutation.

[0385] Preferred primers include those disclosed in Table 13.

[0386] C. Sequencing of Amplified Genomic DNA And Identification ofSingle Nucleotide Polymorphisms

[0387] The amplification products generated as described above, are thensequenced using any method known and available to the skilledtechnician. Methods for sequencing DNA using either the dideoxy-mediatedmethod (Sanger method) or the Maxam-Gilbert method are widely known tothose of ordinary skill in the art. Such methods are for exampledisclosed in Maniatis et al. (Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Press, 2nd Edition, 1989). Alternative approachesinclude hybridization to high-density DNA probe arrays as described inChee et al. (Science 274:610, 1996).

[0388] Preferably, the amplified DNA is subjected to automated dideoxyterminator sequencing reactions using a dye-primer cycle sequencingprotocol. The products of the sequencing reactions are run on sequencinggels and the sequences are determined using gel image analysis. Thepolymorphism search is based on the presence of superimposed peaks inthe electrophoresis pattern resulting from different bases occurring atthe same position. Because each dideoxy terminator is labeled with adifferent fluorescent molecule, the two peaks corresponding to abiallelic site present distinct colors corresponding to two differentnucleotides at the same position on the sequence. However, the presenceof two peaks can be an artifact due to background noise. To exclude suchan artifact, the two DNA strands are sequenced and a comparison betweenthe peaks is carried out. In order to be registered as a polymorphicsequence, the polymorphism has to be detected on both strands.

[0389] The above procedure permits those amplification products, whichcontain biallelic markers to be identified. The detection limit for thefrequency of biallelic polymorphisms detected by sequencing pools of 100individuals is approximately 0.1 for the minor allele, as verified bysequencing pools of known allelic frequencies. However, more than 90% ofthe biallelic polymorphisms detected by the pooling method have afrequency for the minor allele higher than 0.25. Therefore, thebiallelic markers selected by this method have a frequency of at least0.1 for the minor allele and less than 0.9 for the major allele.Preferably at least 0.2 for the minor allele and less than 0.8 for themajor allele, more preferably at least 0.3 for the minor allele and lessthan 0.7 for the major allele, thus a heterozygosity rate higher than0.18, preferably higher than 0.32, more preferably higher than 0.42.

[0390] In another embodiment, biallelic markers are detected bysequencing individual DNA samples, the frequency of the minor allele ofsuch a biallelic marker may be less than 0. 1.

[0391] The markers carried by the same fragment of genomic DNA, such asthe insert in a BAC clone, need not necessarily be ordered with respectto one another within the genomic fragment to conduct associationstudies. However, in some embodiments of the present invention, theorder of biallelic markers carried by the same fragment of genomic DNAare determined.

[0392] D. Validation of the Biallelic Markers of the Present Invention

[0393] The polymorphisms are evaluated for their usefulness as geneticmarkers by validating that both alleles are present in a population.Validation of the biallelic markers is accomplished by genotyping agroup of individuals by a method of the invention and demonstrating thatboth alleles are present. Microsequencing is a preferred method ofgenotyping alleles. The validation by genotyping step may be performedon individual samples derived from each individual in the group or bygenotyping a pooled sample derived from more than one individual. Thegroup can be as small as one individual if that individual isheterozygous for the allele in question. Preferably the group containsat least three individuals, more preferably the group contains five orsix individuals, so that a single validation test will be more likely toresult in the validation of more of the biallelic markers that are beingtested. It should be noted, however, that when the validation test isperformed on a small group it may result in a false negative result ifas a result of sampling error none of the individuals tested carries oneof the two alleles. Thus, the validation process is less useful indemonstrating that a particular initial result is an artifact, than itis at demonstrating that there is a bona fide biallelic marker at aparticular position in a sequence. For an indication of whether aparticular biallelic marker has been validated see Table 7(A-B). All ofthe genotyping, haplotyping, association, and interaction study methodsof the invention may optionally be performed solely with validatedbiallelic markers.

[0394] E. Evaluation of the Frequency of the Biallelic Markers of thePresent Invention

[0395] The validated biallelic markers are further evaluated for theirusefulness as genetic markers by determining the frequency of the leastcommon allele at the biallelic marker site. The determination of theleast common allele is accomplished by genotyping a group of individualsby a method of the invention and demonstrating that both alleles arepresent. This determination of frequency by genotyping step may beperformed on individual samples derived from each individual in thegroup or by genotyping a pooled sample derived from more than oneindividual. The group must be large enough to be representative of thepopulation as a whole. Preferably the group contains at least 20individuals, more preferably the group contains at least 50 individuals,most preferably the group contains at least 100 individuals. Of coursethe larger the group the greater the accuracy of the frequencydetermination because of reduced sampling error. For an indication ofthe frequency for the less common allele of a particular biallelicmarker of the invention see Table 7(A-B). A biallelic marker wherein thefrequency of the less common allele is 30% or more is termed a “highquality biallelic marker.” All of the genotyping, haplotyping,association, and interaction study methods of the invention mayoptionally be performed solely with high quality biallelic markers.

[0396] III. Methods of Genotyping an Individual for Biallelic Markers

[0397] Methods are provided to genotype a biological sample for one ormore biallelic markers of the present invention, all of which may beperformed in vitro. Such methods of genotyping comprise determining theidentity of a nucleotide at an eicosanoid-related biallelic marker byany method known in the art. These methods find use in genotypingcase-control populations in association studies as well as individualsin the context of detection of alleles of biallelic markers which, areknown to be associated with a given trait, in which case both copies ofthe biallelic marker present in individual's genome are determined sothat an individual may be classified as homozygous or heterozygous for aparticular allele.

[0398] These genotyping methods can be performed nucleic acid samplesderived from a single individual or pooled DNA samples.

[0399] Genotyping can be performed using similar methods as thosedescribed above for the identification of the biallelic markers, orusing other genotyping methods such as those further described below. Inpreferred embodiments, the comparison of sequences of amplified genomicfragments from different individuals is used to identify new biallelicmarkers whereas microsequencing is used for genotyping known biallelicmarkers in diagnostic and association study applications.

[0400] A. Source of DNA for Genotyping

[0401] Any source of nucleic acids, in purified or non-purified form,can be utilized as the starting nucleic acid, provided it contains or issuspected of containing the specific nucleic acid sequence desired. DNAor RNA may be extracted from cells, tissues, body fluids and the like asdescribed above in II.A. While nucleic acids for use in the genotypingmethods of the invention can be derived from any mammalian source, thetest subjects and individuals from which nucleic acid samples are takenare generally understood to be human.

[0402] B. Amplification Of DNA Fragments Comprising Biallelic Markers

[0403] Methods and polynucleotides are provided to amplify a segment ofnucleotides comprising one or more biallelic marker of the presentinvention. It will be appreciated that amplification of DNA fragmentscomprising biallelic markers may be used in various methods and forvarious purposes and is not restricted to genotyping. Nevertheless, manygenotyping methods, although not all, require the previous amplificationof the DNA region carrying the biallelic marker of interest. Suchmethods specifically increase the concentration or total number ofsequences that span the biallelic marker or include that site andsequences located either distal or proximal to it. Diagnostic assays mayalso rely on amplification of DNA segments carrying a biallelic markerof the present invention.

[0404] Amplification of DNA may be achieved by any method known in theart. The established PCR (polymerase chain reaction) method or bydevelopments thereof or alternatives. Amplification methods which can beutilized herein include but are not limited to Ligase Chain Reaction(LCR) as described in EP A 320 308 and EP A 439 182, Gap LCR (Wolcott,M. J., Clin. Mcrobiol. Rev. 5:370-386), the so-called “NASBA” or “3SR”technique described in Guatelli J. C. et al. (Proc. Natl. Acad. Sci. USA87:1874-1878, 1990) and in Compton J. (Nature 350:91-92, 1991), Q-betaamplification as described in European Patent Application no 4544610,strand displacement amplification as described in Walker et al. (Clin.Chem. 42:9-13, 1996) and EP A 684 315 and, target mediated amplificationas described in PCT Publication WO 9322461.

[0405] LCR and Gap LCR are exponential amplification techniques, bothdepend on DNA ligase to join adjacent primers annealed to a DNAmolecule. In Ligase Chain Reaction (LCR), probe pairs are used whichinclude two primary (first and second) and two secondary (third andfourth) probes, all of which are employed in molar excess to target. Thefirst probe hybridizes to a first segment of the target strand and thesecond probe hybridizes to a second segment of the target strand, thefirst and second segments being contiguous so that the primary probesabut one another in 5′ phosphate-3′hydroxyl relationship, and so that aligase can covalently fuse or ligate the two probes into a fusedproduct. In addition, a third (secondary) probe can hybridize to aportion of the first probe and a fourth (secondary) probe can hybridizeto a portion of the second probe in a similar abutting fashion. Ofcourse, if the target is initially double stranded, the secondary probesalso will hybridize to the target complement in the first instance. Oncethe ligated strand of primary probes is separated from the targetstrand, it will hybridize with the third and fourth probes which can beligated to form a complementary, secondary ligated product. It isimportant to realize that the ligated products are functionallyequivalent to either the target or its complement. By repeated cycles ofhybridization and ligation, amplification of the target sequence isachieved. A method for multiplex LCR has also been described (WO9320227). Gap LCR (GLCR) is a version of LCR where the probes are notadjacent but are separated by 2 to 3 bases.

[0406] For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR(RT-AGLCR) as described by Marshall R. L. et al. (PCR Methods andApplications 4:80-84, 1994). AGLCR is a modification of GLCR that allowsthe amplification of RNA.

[0407] Some of these amplification methods are particularly suited forthe detection of single nucleotide polymorphisms and allow thesimultaneous amplification of a target sequence and the identificationof the polymorphic nucleotide as it is further described in IIIC.

[0408] The PCR technology is the preferred amplification technique usedin the present invention. A variety of PCR techniques are familiar tothose skilled in the art. For a review of PCR technology, see MolecularCloning to Genetic Engineering White, B. A. Ed. in Methods in MolecularBiology 67: Humana Press, Totowa (1997) and the publication entitled“PCR Methods and Applications” (1991, Cold Spring Harbor LaboratoryPress). In each of these PCR procedures, PCR primers on either side ofthe nucleic acid sequences to be amplified are added to a suitablyprepared nucleic acid sample along with dNTPs and a thermostablepolymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.The nucleic acid in the sample is denatured and the PCR primers arespecifically hybridized to complementary nucleic acid sequences in thesample. The hybridized primers are extended. Thereafter, another cycleof denaturation, hybridization, and extension is initiated. The cyclesare repeated multiple times to produce an amplified fragment containingthe nucleic acid sequence between the primer sites. PCR has further beendescribed in several patents including U.S. Pat. Nos. 4,683,195,4,683,202 and 4,965,188.

[0409] The identification of biallelic markers as described above allowsthe design of appropriate oligonucleotides, which can be used as primersto amplify DNA fragments comprising the biallelic markers of the presentinvention. Amplification can be performed using the primers initiallyused to discover new biallelic markers which are described herein or anyset of primers allowing the amplification of a DNA fragment comprising abiallelic marker of the present invention. Primers can be prepared byany suitable method. As for example, direct chemical synthesis by amethod such as the phosphodiester method of Narang S. A. et al. (MethodsEnzymol. 68:90-98, 1979), the phosphodiester method of Brown E. L. etal. (Methods Enzymol. 68:109-151, 1979), the diethylphosphoramiditemethod of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981) and thesolid support method described in EP 0 707 592.

[0410] In some embodiments the present invention provides primers foramplifying a DNA fragment containing one or more biallelic markers ofthe present invention. Preferred amplification primers are listed inTable 13. It will be appreciated that the primers listed are merelyexemplary and that any other set of primers which produce amplificationproducts containing one or more biallelic markers of the presentinvention.

[0411] The primers are selected to be substantially complementary to thedifferent strands of each specific sequence to be amplified. The lengthof the primers of the present invention can range from 8 to 100nucleotides, preferably from 8 to 50, 8 to 30 or more preferably 8 to 25nucleotides. Shorter primers tend to lack specificity for a targetnucleic acid sequence and generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. Longer primersare expensive to produce and can sometimes self-hybridize to formhairpin structures. The formation of stable hybrids depends on themelting temperature (Tm) of the DNA. The Tm depends on the length of theprimer, the ionic strength of the solution and the G+C content. Thehigher the G+C content of the primer, the higher is the meltingtemperature because G:C pairs are held by three H bonds whereas A:Tpairs have only two. The G+C content of the amplification primers of thepresent invention preferably ranges between 10 and 75%, more preferablybetween 35 and 60%, and most preferably between 40 and 55%. Theappropriate length for primers under a particular set of assayconditions may be empirically determined by one of skill in the art.

[0412] The spacing of the primers determines the length of the segmentto be amplified. In the context of the present invention amplifiedsegments carrying biallelic markers can range in size from at leastabout 25 bp to 35 kbp. Amplification fragments from 25-3000 bp aretypical, fragments from 50-1000 bp are preferred and fragments from100-600 bp are highly preferred. It will be appreciated thatamplification primers for the biallelic markers may be any sequencewhich allow the specific amplification of any DNA fragment carrying themarkers. Amplification primers may be labeled or immobilized on a solidsupport as described in I “Biallelic Markers and PolynucleotidesComprising Biallelic Markers.”

[0413] C. Methods of Genotyping DNA Samples for Biallelic Markers

[0414] Any method known in the art can be used to identify thenucleotide present at a biallelic marker site. Since the biallelicmarker allele to be detected has been identified and specified in thepresent invention, detection will prove simple for one of ordinary skillin the art by employing any of a number of techniques. Many genotypingmethods require the previous amplification of the DNA region carryingthe biallelic marker of interest. While the amplification of target orsignal is often preferred at present, ultrasensitive detection methodswhich do not require amplification are also encompassed by the presentgenotyping methods. Methods well-known to those skilled in the art thatcan be used to detect biallelic polymorphisms include methods such as,conventional dot blot analyzes, single strand conformationalpolymorphism analysis (SSCP) described by Orita et al. (Proc. Natl.Acad. Sci. U.S.A 86:27776-2770, 1989), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis, mismatch cleavagedetection, and other conventional techniques as described in Sheffield,V. C. et al. (Proc. Natl. Acad. Sci. USA 49:699-706, 1991), White et al.(Genomics 12:301-306, 1992), Grompe, M. et al. (Proc. Natl. Acad. Sci.USA 86:5855-5892, 1989) and Grompe, M. (Nature Genetics 5:111-117,1993). Another method for determining the identity of the nucleotidepresent at a particular polymorphic site employs a specializedexonuclease-resistant nucleotide derivative as described in U.S. Pat.No. 4,656,127.

[0415] Preferred methods involve directly determining the identity ofthe nucleotide present at a biallelic marker site by sequencing assay,enzyme-based mismatch detection assay, or hybridization assay. Thefollowing is a description of some preferred methods. A highly preferredmethod is the microsequencing technique. The term “sequencing assay” isused herein to refer to polymerase extension of duplex primer/templatecomplexes and includes both traditional sequencing and microsequencing.

[0416] 1. Sequencing Assays.

[0417] The nucleotide present at a polymorphic site can be determined bysequencing methods. In a preferred embodiment, DNA samples are subjectedto PCR amplification before sequencing as described above. DNAsequencing methods are described in IIC.

[0418] Preferably, the amplified DNA is subjected to automated dideoxyterminator sequencing reactions using a dye-primer cycle sequencingprotocol. Sequence analysis allows the identification of the basepresent at the biallelic marker site.

[0419] 2. Microsequencing Assays

[0420] In microsequencing methods, a nucleotide at the polymorphic sitethat is unique to one of the alleles in a target DNA is detected by asingle nucleotide primer extension reaction. This method involvesappropriate microsequencing primers which, hybridize just upstream of apolymorphic base of interest in the target nucleic acid. A polymerase isused to specifically extend the 3′ end of the primer with one singleddNTP (chain terminator) complementary to the selected nucleotide at thepolymorphic site. Next the identity of the incorporated nucleotide isdetermined in any suitable way.

[0421] Typically, microsequencing reactions are carried out usingfluorescent ddNTPs and the extended microsequencing primers are analyzedby electrophoresis on ABI 377 sequencing machines to determine theidentity of the incorporated nucleotide as described in EP 412 883.Alternatively capillary electrophoresis can be used in order to processa higher number of assays simultaneously. An example of a typicalmicrosequencing procedure that can be used in the context of the presentinvention is provided in Example 2.

[0422] Different approaches can be used to detect the nucleotide addedto the microsequencing primer. A homogeneous phase detection methodbased on fluorescence resonance energy transfer has been described byChen and Kwok (Nucleic Acids Research 25:347-353 1997) and Chen et al.(Proc. Natl. Acad. Sci. USA 94/20 10756-10761,1997). In this methodamplified genomic DNA fragments containing polymorphic sites areincubated with a 5′-fluorescein-labeled primer in the presence ofallelic dye-labeled dideoxyribonucleoside triphosphates and a modifiedTaq polymerase. The dye-labeled primer is extended one base by thedye-terminator specific for the allele present on the template. At theend of the genotyping reaction, the fluorescence intensities of the twodyes in the reaction mixture are analyzed directly without separation orpurification. All these steps can be performed in the same tube and thefluorescence changes can be monitored in real time. Alternatively, theextended primer may be analyzed by MALDI-TOF Mass Spectrometry. The baseat the polymorphic site is identified by the mass added onto themicrosequencing primer (see Haff L. A. and Smirnov I. P., GenomeResearch, 7:378-388, 1997).

[0423] Microsequencing may be achieved by the establishedmicrosequencing method or by developments or derivatives thereof.Alternative methods include several solid-phase microsequencingtechniques. The basic microsequencing protocol is the same as describedpreviously, except that the method is conducted as a heterogenous phaseassay, in which the primer or the target molecule is immobilized orcaptured onto a solid support. To simplify the primer separation and theterminal nucleotide addition analysis, oligonucleotides are attached tosolid supports or are modified in such ways that permit affinityseparation as well as polymerase extension. The 5′ ends and internalnucleotides of synthetic oligonucleotides can be modified in a number ofdifferent ways to permit different affinity separation approaches, e.g.,biotinylation. If a single affinity group is used on theoligonucleotides, the oligonucleotides can be separated from theincorporated terminator reagent. This eliminates the need of physical orsize separation. More than one oligonucleotide can be separated from theterminator reagent and analyzed simultaneously if more than one affinitygroup is used. This permits the analysis of several nucleic acid speciesor more nucleic acid sequence information per extension reaction. Theaffinity group need not be on the priming oligonucleotide but couldalternatively be present on the template. For example, immobilizationcan be carried out via an interaction between biotinylated DNA andstreptavidin-coated microtitration wells or avidin-coated polystyreneparticles. In the same manner oligonucleotides or templates may beattached to a solid support in a high-density format. In such solidphase microsequencing reactions, incorporated ddNTPs can be radiolabeled(Syvänen, Clinica Chimica Acta 226:225-236, 1994) or linked tofluorescein (Livak and Hainer, Human Mutation 3:379-385,1994). Thedetection of radiolabeled ddNTPs can be achieved throughscintillation-based techniques. The detection of fluorescein-linkedddNTPs can be based on the binding of antifluorescein antibodyconjugated with alkaline phosphatase, followed by incubation with achromogenic substrate (such as p-nitrophenyl phosphate). Other possiblereporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP)and anti-DNP alkaline phosphatase conjugate (Harju et al., Clin. Chem.39/11 2282-2287, 1993) or biotinylated ddNTP and horseradishperoxidase-conjugated streptavidin with o-phenylenediamine as asubstrate (WO 92/15712). As yet another alternative solid-phasemicrosequencing procedure, Nyren et al. (Analytical Biochemistry 208:171-175, 1993) described a method relying on the detection of DNApolymerase activity by an enzymatic luminometric inorganic pyrophosphatedetection assay (ELIDA).

[0424] Pastinen et al. (Genome research 7:606-614, 1997) describe amethod for multiplex detection of single nucleotide polymorphism inwhich the solid phase minisequencing principle is applied to anoligonucleotide array format. High-density arrays of DNA probes attachedto a solid support (DNA chips) are further described in III.C.5.

[0425] In one aspect the present invention provides polynucleotides andmethods to genotype one or more biallelic markers of the presentinvention by performing a microsequencing assay. Preferredmicrosequencing primers include those being featured Table 12. It willbe appreciated that the microsequencing primers listed in Table 12 aremerely exemplary and that, any primer having a 3′ end immediatelyadjacent to a polymorphic nucleotide may be used. Similarly, it will beappreciated that microsequencing analysis may be performed for anybiallelic marker or any combination of biallelic markers of the presentinvention. One aspect of the present invention is a solid support whichincludes one or more microsequencing primers listed in Table 12, orfragments comprising at least 8, at least 12, at least 15, or at least20 consecutive nucleotides thereof and having a 3′ terminus immediatelyupstream of the corresponding biallelic marker, for determining theidentity of a nucleotide at biallelic marker site.

[0426] 3. Mismatch Detection Assays Based on Polymerases and Ligases

[0427] In one aspect the present invention provides polynucleotides andmethods to determine the allele of one or more biallelic markers of thepresent invention in a biological sample, by mismatch detection assaysbased on polymerases and/or ligases. These assays are based on thespecificity of polymerases and ligases. Polymerization reactions placesparticularly stringent requirements on correct base pairing of the 3′end of the amplification primer and the joining of two oligonucleotideshybridized to a target DNA sequence is quite sensitive to mismatchesclose to the ligation site, especially at the 3′ end. The terms “enzymebased mismatch detection assay” are used herein to refer to any methodof determining the allele of a biallelic marker based on the specificityof ligases and polymerases. Preferred methods are described below.Methods, primers and various parameters to amplify DNA fragmentscomprising biallelic markers of the present invention are furtherdescribed above in III.B.

[0428] Allele Specific Amplification

[0429] Discrimination between the two alleles of a biallelic marker canalso be achieved by allele specific amplification, a selective strategy,whereby one of the alleles is amplified without amplification of theother allele. This is accomplished by placing a polymorphic base at the3′ end of one of the amplification primers. Because the extension formsfrom the 3′end of the primer, a mismatch at or near this position has aninhibitory effect on amplification. Therefore, under appropriateamplification conditions, these primers only direct amplification ontheir complementary allele. Designing the appropriate allele-specificprimer and the corresponding assay conditions are well with the ordinaryskill in the art.

[0430] Ligation/Amplification Based Methods

[0431] The “Oligonucleotide Ligation Assay” (OLA) uses twooligonucleotides which are designed to be capable of hybridizing toabutting sequences of a single strand of a target molecules. One of theoligonucleotides is biotinylated, and the other is detectably labeled.If the precise complementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting biallelic markers and may be advantageously combined withPCR as described by Nickerson D. A. et al. (Proc. Natl. Acad. Sci.U.S.A. 87:8923-8927, 1990). In this method, PCR is used to achieve theexponential amplification of target DNA, which is then detected usingOLA.

[0432] Other methods which are particularly suited for the detection ofbiallelic markers include LCR (ligase chain reaction), Gap LCR (GLCR)which are described above in III.B. As mentioned above LCR uses twopairs of probes to exponentially amplify a specific target. Thesequences of each pair of oligonucleotides, is selected to permit thepair to hybridize to abutting sequences of the same strand of thetarget. Such hybridization forms a substrate for a template-dependantligase. In accordance with the present invention, LCR can be performedwith oligonucleotides having the proximal and distal sequences of thesame strand of a biallelic marker site. In one embodiment, eitheroligonucleotide will be designed to include the biallelic marker site.In such an embodiment, the reaction conditions are selected such thatthe oligonucleotides can be ligated together only if the target moleculeeither contains or lacks the specific nucleotide(s) that iscomplementary to the biallelic marker on the oligonucleotide. In analternative embodiment, the oligonucleotides will not include thebiallelic marker, such that when they hybridize to the target molecule,a “gap” is created as described in WO 90/01069. This gap is then“filled” with complementary dNTPs (as mediated by DNA polymerase), or byan additional pair of oligonucleotides. Thus at the end of each cycle,each single strand has a complement capable of serving as a targetduring the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained.

[0433] Ligase/Polymerase-mediated Genetic Bit Analysis™ is anothermethod for determining the identity of a nucleotide at a preselectedsite in a nucleic acid molecule (WO 95/21271). This method involves theincorporation of a nucleoside triphosphate that is complementary to thenucleotide present at the preselected site onto the terminus of a primermolecule, and their subsequent ligation to a second oligonucleotide. Thereaction is monitored by detecting a specific label attached to thereaction's solid phase or by detection in solution.

[0434] 4. Hybridization Assay Methods

[0435] A preferred method of determining the identity of the nucleotidepresent at a biallelic marker site involves nucleic acid hybridization.The hybridization probes, which can be conveniently used in suchreactions, preferably include the probes defined herein. Anyhybridization assay may be used including Southern hybridization,Northern hybridization, dot blot hybridization and solid-phasehybridization (see Sambrook et al., Molecular Cloning—A LaboratoryManual, Second Edition, Cold Spring Harbor Press, N.Y., 1989).

[0436] Hybridization refers to the formation of a duplex structure bytwo single stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch. Specific probes can be designed that hybridize to one form ofa biallelic marker and not to the other and therefore are able todiscriminate between different allelic forms. Allele-specific probes areoften used in pairs, one member of a pair showing perfect match to atarget sequence containing the original allele and the other showing aperfect match to the target sequence containing the alternative allele.Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Stringent, sequence specific hybridizationconditions, under which a probe will hybridize only to the exactlycomplementary target sequence are well known in the art (Sambrook etal., Molecular Cloning—A Laboratory Manual, Second Edition, Cold SpringHarbor Press, N.Y., 1989). Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.By way of example and not limitation, procedures using conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C., the preferred hybridization temperature,in prehybridization mixture containing 100 μg/ml denatured salmon spermDNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, thehybridization step can be performed at 65° C. in the presence of SSCbuffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals.Following the wash steps, the hybridized probes are detectable byautoradiography. By way of example and not limitation, procedures usingconditions of intermediate stringency are as follows: Filters containingDNA are prehybridized, and then hybridized at a temperature of 60° C. inthe presence of a 5×SSC buffer and labeled probe. Subsequently, filterswashes are performed in a solution containing 2×SSC at 50° C. and thehybridized probes are detectable by autoradiography. Other conditions ofhigh and intermediate stringency which may be used are well known in theart and as cited in Sambrook et al. (Molecular Cloning—A LaboratoryManual, Second Edition, Cold Spring Harbor Press, N.Y., 1989) andAusubel et al. (Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y., 1989).

[0437] Although such hybridizations can be performed in solution, it ispreferred to employ a solid-phase hybridization assay. The target DNAcomprising a biallelic marker of the present invention may be amplifiedprior to the hybridization reaction. The presence of a specific allelein the sample is determined by detecting the presence or the absence ofstable hybrid duplexes formed between the probe and the target DNA. Thedetection of hybrid duplexes can be carried out by a number of methods.Various detection assay formats are well known which utilize detectablelabels bound to either the target or the probe to enable detection ofthe hybrid duplexes. Typically, hybridization duplexes are separatedfrom unhybridized nucleic acids and the labels bound to the duplexes arethen detected. Those skilled in the art will recognize that wash stepsmay be employed to wash away excess target DNA or probe. Standardheterogeneous assay formats are suitable for detecting the hybrids usingthe labels present on the primers and probes.

[0438] Two recently developed assays allow hybridization-based allelediscrimination with no need for separations or washes (see Landegren U.et al., Genome Research, 8:769-776,1998). The TaqMan assay takesadvantage of the 5′ nuclease activity of Taq DNA polymerase to digest aDNA probe annealed specifically to the accumulating amplificationproduct. TaqMan probes are labeled with a donor-acceptor dye pair thatinteracts via fluorescence energy transfer. Cleavage of the TaqMan probeby the advancing polymerase during amplification dissociates the donordye from the quenching acceptor dye, greatly increasing the donorfluorescence. All reagents necessary to detect two allelic variants canbe assembled at the beginning of the reaction and the results aremonitored in real time (see Livak et al., Nature Genetics, 9:341-342,1995). In an alternative homogeneous hybridization-based procedure,molecular beacons are used for allele discriminations. Molecular beaconsare hairpin-shaped oligonucleotide probes that report the presence ofspecific nucleic acids in homogeneous solutions. When they bind to theirtargets they undergo a conformational reorganization that restores thefluorescence of an internally quenched fluorophore (Tyagi et al., NatureBiotechnology, 16:49-53, 1998).

[0439] The polynucleotides provided herein can be used in hybridizationassays for the detection of biallelic marker alleles in biologicalsamples. These probes are characterized in that they preferably comprisebetween 8 and 50 nucleotides, and in that they are sufficientlycomplementary to a sequence comprising a biallelic marker of the presentinvention to hybridize thereto and preferably sufficiently specific tobe able to discriminate the targeted sequence for only one nucleotidevariation. The GC content in the probes of the invention usually rangesbetween 10 and 75%, preferably between 35 and 60%, and more preferablybetween 40 and 55%. The length of these probes can range from 10, 15,20, or 30 to at least 100 nucleotides, preferably from 10 to 50, morepreferably from 18 to 35 nucleotides. A particularly preferred probe is25 nucleotides in length. Preferably the biallelic marker is within 4nucleotides of the center of the polynucleotide probe. In particularlypreferred probes the biallelic marker is at the center of saidpolynucleotide. Shorter probes may lack specificity for a target nucleicacid sequence and generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. Longer probesare expensive to produce and can sometimes self-hybridize to formhairpin structures. Methods for the synthesis of oligonucleotide probeshave been described above and can be applied to the probes of thepresent invention.

[0440] Preferably the probes of the present invention are labeled orimmobilized on a solid support. Labels and solid supports are furtherdescribed in I. Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example peptide nucleicacids which are disclosed in International Patent Application WO92/20702, morpholino analogs which are described in U.S. Pat. Nos.5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered“non-extendable” in that additional dNTPs cannot be added to the probe.In and of themselves analogs usually are non-extendable and nucleic acidprobes can be rendered non-extendable by modifying the 3′ end of theprobe such that the hydroxyl group is no longer capable of participatingin elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified, U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 describes modifications, whichcan be used to render a probe non-extendable.

[0441] The probes of the present invention are useful for a number ofpurposes. They can be used in Southern hybridization to genomic DNA orNorthern hybridization to mRNA. The probes can also be used to detectPCR amplification products. By assaying the hybridization to an allelespecific probe, one can detect the presence or absence of a biallelicmarker allele in a given sample.

[0442] High-Throughput parallel hybridizations in array format arespecifically encompassed within “hybridization assays” and are describedbelow.

[0443] Hybridization to Addressable Arrays of Oligonucleotides

[0444] Hybridization assays based on oligonucleotide arrays rely on thedifferences in hybridization stability of short oligonucleotides toperfectly matched and mismatched target sequence variants. Efficientaccess to polymorphism information is obtained through a basic structurecomprising high-density arrays of oligonucleotide probes attached to asolid support (the chip) at selected positions. Each DNA chip cancontain thousands to millions of individual synthetic DNA probesarranged in a grid-like pattern and miniaturized to the size of a dime.

[0445] The chip technology has already been applied with success innumerous cases. For example, the screening of mutations has beenundertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and inthe protease gene of HIV-1 virus (Hacia et al., Nature Genetics,14(4):441-447, 1996; Shoemaker et al., Nature Genetics, 14(4):450-456,1996; Kozal et al., Nature Medicine, 2:753-759, 1996). Chips of variousformats for use in detecting biallelic polymorphisms can be produced ona customized basis by Affymetrix (GeneChip™), Hyseq (HyChip andHyGnostics), and Protogene Laboratories.

[0446] In general, these methods employ arrays of oligonucleotide probesthat are complementary to target nucleic acid sequence segments from anindividual which, target sequences include a polymorphic marker.EP785280 describes a tiling strategy for the detection of singlenucleotide polymorphisms. Briefly, arrays may generally be “tiled” for alarge number of specific polymorphisms. By “tiling” is generally meantthe synthesis of a defined set of oligonucleotide probes which is madeup of a sequence complementary to the target sequence of interest, aswell as preselected variations of that sequence, e.g., substitution ofone or more given positions with one or more members of the basis set ofmonomers, i.e. nucleotides. Tiling strategies are further described inPCT application No. WO 95/11995. In a particular aspect, arrays aretiled for a number of specific, identified biallelic marker sequences.In particular the array is tiled to include a number of detectionblocks, each detection block being specific for a specific biallelicmarker or a set of biallelic markers. For example, a detection block maybe tiled to include a number of probes, which span the sequence segmentthat includes a specific polymorphism. To ensure probes that arecomplementary to each allele, the probes are synthesized in pairsdiffering at the biallelic marker. In addition to the probes differingat the polymorphic base, monosubstituted probes are also generally tiledwithin the detection block. These monosubstituted probes have bases atand up to a certain number of bases in either direction from thepolymorphism, substituted with the remaining nucleotides (selected fromA, T, G, C and U). Typically the probes in a tiled detection block willinclude substitutions of the sequence positions up to and includingthose that are 5 bases away from the biallelic marker. Themonosubstituted probes provide internal controls for the tiled array, todistinguish actual hybridization from artefactual cross-hybridization.Upon completion of hybridization with the target sequence and washing ofthe array, the array is scanned to determine the position on the arrayto which the target sequence hybridizes. The hybridization data from thescanned array is then analyzed to identify which allele or alleles ofthe biallelic marker are present in the sample. Hybridization andscanning may be carried out as described in PCT application No. WO92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186.

[0447] Thus, in some embodiments, the chips may comprise an array ofnucleic acid sequences of fragments of about 15 nucleotides in length.In further embodiments, the chip may comprise an array including atleast one of the sequences selected from the group consisting of SEQ IDNos. 1-70, 72-654 except SEQ ID Nos. 419-424, 490, 531 and 540 and thesequences complementary thereto, or more preferably SEQ ID Nos. 655-724,726-1304 except SEQ ID Nos. 1073-1078, 1144, 1185, 1194 and thesequences complementary thereto, or a fragment thereof at least about 8consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30,40, 47, or 50 consecutive nucleotides. In some embodiments, the chip maycomprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of thesepolynucleotides of the invention. Solid supports and polynucleotides ofthe present invention attached to solid supports are further describedin I. Biallelic Markers and Polynucleotides Comprising BiallelicMarkers.

[0448] 5. Integrated Systems

[0449] Another technique, which may be used to analyze polymorphisms,includes multicomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device. An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, which describes the integration ofPCR amplification and capillary electrophoresis in chips.

[0450] Integrated systems can be envisaged mainly when micro fluidicsystems are used. These systems comprise a pattern of micro channelsdesigned onto a glass, silicon, quartz, or plastic wafer included on amicrochip. The movements of the samples are controlled by electric,electroosmotic or hydrostatic forces applied across different areas ofthe microchip. For genotyping biallelic markers, the microfluidic systemmay integrate nucleic acid amplification, microsequencing, capillaryelectrophoresis and a detection method such as laser-inducedfluorescence detection.

[0451] IV. Methods of Genetic Analysis Using the Biallelic Markers ofthe Present Invention

[0452] Different methods are available for the genetic analysis ofcomplex traits (see Lander and Schork, Science, 265, 2037-2048, 1994).The search for disease-susceptibility genes is conducted using two mainmethods: the linkage approach in which evidence is sought forcosegregation between a locus and a putative trait locus using familystudies, and the association approach in which evidence is sought for astatistically significant association between an allele and a trait or atrait causing allele (Khoury J. et al., Fundamentals of GeneticEpidemiology, Oxford University Press, NY, 1993). In general, thebiallelic markers of the present invention find use in any method knownin the art to demonstrate a statistically significant correlationbetween a genotype and a phenotype. The biallelic markers may be used inparametric and non-parametric linkage analysis methods. Preferably, thebiallelic markers of the present invention are used to identify genesassociated with detectable traits using association studies, an approachwhich does not require the use of affected families and which permitsthe identification of genes associated with complex and sporadic traits.

[0453] The genetic analysis using the biallelic markers of the presentinvention may be conducted on any scale. The whole set of biallelicmarkers of the present invention or any subset of biallelic markers ofthe present invention may be used. In some embodiments a subset ofbiallelic markers corresponding to one or several candidate genes of thepresent invention may be used. In other embodiments a subset ofbiallelic markers corresponding to candidate genes from a given pathwayof arachidonic acid metabolism may be used. Such pathways include thecycloxygenase pathway and the lipoxygenase pathway. Alternatively, asubset of biallelic markers of the present invention localised on aspecific chromosome segment may be used. Further, any set of geneticmarkers including a biallelic marker of the present invention may beused. A set of biallelic polymorphisms that, could be used as geneticmarkers in combination with the biallelic markers of the presentinvention, has been described in WO 98/20165. As mentioned above, itshould be noted that the biallelic markers of the present invention maybe included in any complete or partial genetic map of the human genome.These different uses are specifically contemplated in the presentinvention and claims.

[0454] A. Linkage Analysis

[0455] Linkage analysis is based upon establishing a correlation betweenthe transmission of genetic markers and that of a specific traitthroughout generations within a family. Thus, the aim of linkageanalysis is to detect marker loci that show cosegregation with a traitof interest in pedigrees.

[0456] Parametric Methods

[0457] When data are available from successive generations there is theopportunity to study the degree of linkage between pairs of loci.Estimates of the recombination fraction enable loci to be ordered andplaced onto a genetic map. With loci that are genetic markers, a geneticmap can be established, and then the strength of linkage between markersand traits can be calculated and used to indicate the relative positionsof markers and genes affecting those traits (Weir, B. S., Genetic dataAnalysis II: Methods for Discrete population genetic Data, SinauerAssoc., Inc., Sunderland, Mass., USA, 1996). The classical method forlinkage analysis is the logarithm of odds (lod) score method (see MortonN. E., Am.J. Hum. Genet., 7:277-318, 1955; Ott J., Analysis of HumanGenetic Linkage, John Hopkins University Press, Baltimore, 1991).Calculation of lod scores requires specification of the mode ofinheritance for the disease (parametric method). Generally, the lengthof the candidate region identified using linkage analysis is between 2and 20 Mb. Once a candidate region is identified as described above,analysis of recombinant individuals using additional markers allowsfurther delineation of the candidate region. Linkage analysis studieshave generally relied on the use of a maximum of 5,000 microsatellitemarkers, thus limiting the maximum theoretical attainable resolution oflinkage analysis to about 600 kb on average.

[0458] Linkage analysis has been successfully applied to map simplegenetic traits that show clear Mendelian inheritance patterns and whichhave a high penetrance (i.e., the ratio between the number of affectedcarriers of allele a and the total number of a carriers in thepopulation). However, parametric linkage analysis suffers from a varietyof drawbacks. First, it is limited by its reliance on the choice of agenetic model suitable for each studied trait. Furthermore, as alreadymentioned, the resolution attainable using linkage analysis is limited,and complementary studies are required to refine the analysis of thetypical 2 Mb to 20 Mb regions initially identified through linkageanalysis. In addition, parametric linkage analysis approaches haveproven difficult when applied to complex genetic traits, such as thosedue to the combined action of multiple genes and/or environmentalfactors. It is very difficult to model these factors adequately in a lodscore analysis. In such cases, too large an effort and cost are neededto recruit the adequate number of affected families required forapplying linkage analysis to these situations, as recently discussed byRisch, N. and Merikangas, K. (Science, 273:1516-1517, 1996).

[0459] Non-Parametric Methods

[0460] The advantage of the so-called non-parametric methods for linkageanalysis is that they do not require specification of the mode ofinheritance for the disease, they tend to be more useful for theanalysis of complex traits. In non-parametric methods, one tries toprove that the inheritance pattern of a chromosomal region is notconsistent with random Mendelian segregation by showing that affectedrelatives inherit identical copies of the region more often thanexpected by chance. Affected relatives should show excess “allelesharing” even in the presence of incomplete penetrance and polygenicinheritance. In non-parametric linkage analysis the degree of agreementat a marker locus in two individuals can be measured either by thenumber of alleles identical by state (IBS) or by the number of allelesidentical by descent (IBD). Affected sib pair analysis is a well-knownspecial case and is the simplest form of these methods.

[0461] The biallelic markers of the present invention may be used inboth parametric and non-parametric linkage analysis. Preferablybiallelic markers may be used in non-parametric methods which allow themapping of genes involved in complex traits. The biallelic markers ofthe present invention may be used in both IBD- and IBS- methods to mapgenes affecting a complex trait. In such studies, taking advantage ofthe high density of biallelic markers, several adjacent biallelic markerloci may be pooled to achieve the efficiency attained by multi-allelicmarkers (Zhao et al., Am. J. Hum. Genet., 63:225-240, 1998).

[0462] However, both parametric and non-parametric linkage analysismethods analyse affected relatives, they tend to be of limited value inthe genetic analysis of drug responses or in the analysis of sideeffects to treatments. This type of analysis is impractical in suchcases due to the lack of availability of familial cases. In fact, thelikelihood of having more than one individual in a family being exposedto the same drug at the same time is extremely low.

[0463] B. Population Association Studies

[0464] The present invention comprises methods for identifying one orseveral genes among a set of candidate genes that are associated with adetectable trait using the biallelic markers of the present invention.In one embodiment the present invention comprises methods to detect anassociation between a biallelic marker allele or a biallelic markerhaplotype and a trait. Further, the invention comprises methods toidentify a trait causing allele in linkage disequilibrium with anybiallelic marker allele of the present invention.

[0465] As described above, alternative approaches can be employed toperform association studies: genome-wide association studies, candidateregion association studies and candidate gene association studies. In apreferred embodiment, the biallelic markers of the present invention areused to perform candidate gene association studies. The candidate geneanalysis clearly provides a short-cut approach to the identification ofgenes and gene polymorphisms related to a particular trait when someinformation concerning the biology of the trait is available. Further,the biallelic markers of the present invention may be incorporated inany map of genetic markers of the human genome in order to performgenome-wide association studies. Methods to generate a high-density mapof biallelic markers have been described in WIPO Patent applicationserial number PCT/IB98/01193. The biallelic markers of the presentinvention may further be incorporated in any map of a specific candidateregion of the genome (a specific chromosome or a specific chromosomalsegment for example).

[0466] As mentioned above, association studies may be conducted withinthe general population and are not limited to studies performed onrelated individuals in affected families. Association studies areextremely valuable as they permit the analysis of sporadic ormultifactor traits. Moreover, association studies represent a powerfulmethod for fine-scale mapping enabling much finer mapping of traitcausing alleles than linkage studies. Studies based on pedigrees oftenonly narrow the location of the trait causing allele. Associationstudies using the biallelic markers of the present invention cantherefore be used to refine the location of a trait causing allele in acandidate region identified by Linkage Analysis methods. Moreover, oncea chromosome segment of interest has been identified, the presence of acandidate gene such as a candidate gene of the present invention, in theregion of interest can provide a shortcut to the identification of thetrait causing allele. Biallelic markers of the present invention can beused to demonstrate that a candidate gene is associated with a trait.Such uses are specifically contemplated in the present invention andclaims.

[0467] 1. Determining the Frequency of a Biallelic Marker Allele or of aBiallelic Marker Haplotype in a Population

[0468] Association studies explore the relationships among frequenciesfor sets of alleles between loci. In addition, the present inventionprovides methods of determining the frequency in a population of anallele of a 12-LO-related biallelic marker comprising: a) genotypingindividuals from said population for said biallelic marker and, b)determining the proportional representation of said biallelic marker insaid population. Optionally, said 12-LO-related biallelic marker isselected from the biallelic markers described in Table 2(a-c). Thepresent invention further provides methods of estimating the frequencyof a haplotype for a set of biallelic markers in a population,comprising: a) genotyping each individual in said population for atleast one 12-LO-related biallelic marker; b) genotyping each individualin said population for a second biallelic marker by determining theidentity of the nucleotides at said second biallelic marker for bothcopies of said second biallelic marker present in the genome; and c)applying a haplotype determination method to the identities of thenucleotides determined in steps a) and b) to obtain an estimate of saidfrequency. Optionally, said haplotype determination method is selectedfrom asymmetric PCR amplification, double PCR amplification of specificalleles, the Clark method, or an expectation maximization algorithm.Optionally, said 12-LO-related biallelic marker is selected from thebiallelic markers described in Table 2(a-c).

[0469] Determining the Frequency of an Allele in a Population

[0470] Allelic frequencies of the biallelic markers in a population canbe determined using one of the methods described above under the heading“Methods for genotyping an individual for biallelic markers,” or anygenotyping procedure suitable for this intended purpose. Genotypingpooled samples or individual samples can determine the frequency of abiallelic marker allele in a population. One way to reduce the number ofgenotypings required is to use pooled samples. A major obstacle in usingpooled samples is in terms of accuracy and reproducibility fordetermining accurate DNA concentrations in setting up the pools.Genotyping individual samples provides higher sensitivity,reproducibility and accuracy and; is the preferred method used in thepresent invention. Preferably, each individual is genotyped separatelyand simple gene counting is applied to determine the frequency of anallele of a biallelic marker or of a genotype in a given population.

[0471] Determining the Frequency of a Haplotype in a Population

[0472] The gametic phase of haplotypes is unknown when diploidindividuals are heterozygous at more than one locus. Using genealogicalinformation in families gametic phase can sometimes be inferred (Perlinet al., Am. J Hum. Genet., 55:777-787, 1994). When no genealogicalinformation is available different strategies may be used. Onepossibility is that the multiple-site heterozygous diploids can beeliminated from the analysis, keeping only the homozygotes and thesingle-site heterozygote individuals, but this approach might lead to apossible bias in the sample composition and the underestimation oflow-frequency haplotypes. Another possibility is that single chromosomescan be studied independently, for example, by asymmetric PCRamplification (see Newton et al., Nucleic Acids Res., 17:2503-2516,1989; Wu et al., Proc. Natl. Acad. Sci. USA, 86:2757, 1989) or byisolation of single chromosome by limit dilution followed by PCRamplification (see Ruano et al., Proc. Natl. Acad. Sci. USA,87:6296-6300, 1990). Further, a sample may be haplotyped forsufficiently close biallelic markers by double PCR amplification ofspecific alleles (Sarkar, G. and Sommer S. S., Biotechniques, 1991).These approaches are not entirely satisfying either because of theirtechnical complexity, the additional cost they entail, their lack ofgeneralisation at a large scale, or the possible biases they introduce.To overcome these difficulties, an algorithm to infer the phase ofPCR-amplified DNA genotypes introduced by Clark A. G. (Mol. Biol. Evol.,7:111-122, 1990) may be used. Briefly, the principle is to start fillinga preliminary list of haplotypes present in the sample by examiningunambiguous individuals, that is, the complete homozygotes and thesingle-site heterozygotes. Then other individuals in the same sample arescreened for the possible occurrence of previously recognisedhaplotypes. For each positive identification, the complementaryhaplotype is added to the list of recognised haplotypes, until the phaseinformation for all individuals is either resolved or identified asunresolved. This method assigns a single haplotype to eachmultiheterozygous individual, whereas several haplotypes are possiblewhen there are more than one heterozygous site. Alternatively, one canuse methods estimating haplotype frequencies in a population withoutassigning haplotypes to each individual. Preferably, a method based onan expectation-maximization (EM) algorithm (Dempster et al., J. R. Stat.Soc., 39B: 1-38, 1977) leading to maximum-likelihood estimates ofhaplotype frequencies under the assumption of Hardy-Weinberg proportions(random mating) is used (see Excoffier L. and Slatkin M., Mol. Biol.Evol., 12(5): 921-927, 1995). The EM algorithm is a generalisediterative maximum-likelihood approach to estimation that is useful whendata are ambiguous and/or incomplete. The EM algorithm is used toresolve heterozygotes into haplotypes. Haplotype estimations are furtherdescribed below under the heading “Statistical methods”. Any othermethod known in the art to determine or to estimate the frequency of ahaplotype in a population may also be used.

[0473] 2. Linkage Disequilibrium Analysis.

[0474] Linkage disequilibrium is the non-random association of allelesat two or more loci and represents a powerful tool for mapping genesinvolved in disease traits (see Ajioka R. S. et al., Am. J. Hum. Genet.,60:1439-1447, 1997). Biallelic markers, because they are densely spacedin the human genome and can be genotyped in more numerous numbers thanother types of genetic markers (such as RFLP or VNTR markers), areparticularly useful in genetic analysis based on linkage disequilibrium.The biallelic markers of the present invention may be used in anylinkage disequilibrium analysis method known in the art.

[0475] When a disease mutation is first introduced into a population (bya new mutation or the immigration of a mutation carrier), it necessarilyresides on a single chromosome and thus on a single “background” or“ancestral” haplotype of linked markers. Consequently, there is completedisequilibrium between these markers and the disease mutation: one findsthe disease mutation only in the presence of a specific set of markeralleles. Through subsequent generations recombinations occur between thedisease mutation and these marker polymorphisms, and the disequilibriumgradually dissipates. The pace of this dissipation is a function of therecombination frequency, so the markers closest to the disease gene willmanifest higher levels of disequilibrium than those that are furtheraway. When not broken up by recombination, “ancestral” haplotypes andlinkage disequilibrium between marker alleles at different loci can betracked not only through pedigrees but also through populations. Linkagedisequilibrium is usually seen as an association between one specificallele at one locus and another specific allele at a second locus.

[0476] The pattern or curve of disequilibrium between disease and markerloci is expected to exhibit a maximum that occurs at the disease locus.Consequently, the amount of linkage disequilibrium between a diseaseallele and closely linked genetic markers may yield valuable informationregarding the location of the disease gene. For fine-scale mapping of adisease locus, it is useful to have some knowledge of the patterns oflinkage disequilibrium that exist between markers in the studied region.As mentioned above the mapping resolution achieved through the analysisof linkage disequilibrium is much higher than that of linkage studies.The high density of biallelic markers combined with linkagedisequilibrium analysis provides powerful tools for fine-scale mapping.Different methods to calculate linkage disequilibrium are describedbelow under the heading “Statistical Methods”.

[0477] 3. Population-Based Case-Control Studies of Trait-MarkerAssociations.

[0478] As mentioned above, the occurrence of pairs of specific allelesat different loci on the same chromosome is not random and the deviationfrom random is called linkage disequilibrium. Association studies focuson population frequencies and rely on the phenomenon of linkagedisequilibrium. If a specific allele in a given gene is directlyinvolved in causing a particular trait, its frequency will bestatistically increased in an affected (affected) population, whencompared to the frequency in a trait negative population or in a randomcontrol population. As a consequence of the existence of linkagedisequilibrium, the frequency of all other alleles present in thehaplotype carrying the trait-causing allele will also be increased inaffected (affected) individuals compared to trait negative individualsor random controls. Therefore, association between the trait and anyallele (specifically a biallelic marker allele) in linkagedisequilibrium with the trait-causing allele will suffice to suggest thepresence of a trait-related gene in that particular region. Case-controlpopulations can be genotyped for biallelic markers to identifyassociations that narrowly locate a trait causing allele. As any markerin linkage disequilibrium with one given marker associated with a traitwill be associated with the trait. Linkage disequilibrium allows therelative frequencies in case-control populations of a limited number ofgenetic polymorphisms (specifically biallelic markers) to be analysed asan alternative to screening all possible functional polymorphisms inorder to find trait-causing alleles. Association studies compare thefrequency of marker alleles in unrelated case-control populations, andrepresent powerful tools for the dissection of complex traits.

[0479] Case-Control Populations (Inclusion Criteria)

[0480] Population-based association studies do not concern familialinheritance but compare the prevalence of a particular genetic marker,or a set of markers, in case-control populations. They are case-controlstudies based on comparison of unrelated case (affected or affected)individuals and unrelated control (unaffected or trait negative orrandom) individuals. Preferably the control group is composed ofunaffected or trait negative individuals. Further, the control group isethnically matched to the case population. Moreover, the control groupis preferably matched to the case-population for the main knownconfusion factor for the trait under study (for example age-matched foran age-dependent trait). Ideally, individuals in the two samples arepaired in such a way that they are expected to differ only in theirdisease status. In the following “affected population”, “casepopulation” and “affected population” are used interchangeably.

[0481] An important step in the dissection of complex traits usingassociation studies is the choice of case-control populations (seeLander and Schork, Science, 265, 2037-2048, 1994). A major step in thechoice of case-control populations is the clinical definition of a giventrait or phenotype. Any genetic trait may be analysed by the associationmethod proposed here by carefully selecting the individuals to beincluded in the affected and control phenotypic groups. Four criteriaare often useful: clinical phenotype, age at onset, family history andseverity. The selection procedure for continuous or quantitative traits(such as blood pressure for example) involves selecting individuals atopposite ends of the phenotype distribution of the trait under study, soas to include in these affected and control individuals withnon-overlapping phenotypes. Preferably, case-control populations consistof phenotypically homogeneous populations. Affected and controlpopulations consist of phenotypically uniform populations of individualsrepresenting each between 1 and 98%, preferably between 1 and 80%, morepreferably between 1 and 50%, and more preferably between 1 and 30%,most preferably between 1 and 20% of the total population under study,and selected among individuals exhibiting non-overlapping phenotypes.The clearer the difference between the two trait phenotypes, the greaterthe probability of detecting an association with biallelic markers. Theselection of those drastically different but relatively uniformphenotypes enables efficient comparisons in association studies and thepossible detection of marked differences at the genetic level, providedthat the sample sizes of the populations under study are significantenough.

[0482] In preferred embodiments, a first group of between 50 and 300affected individuals, preferably about 100 individuals, are recruitedaccording to their phenotypes. A similar number of trait negativeindividuals are included in such studies.

[0483] In the present invention, typical examples of inclusion criteriainclude a disease involving arachidonic acid metabolism or theevaluation of the response to a drug acting on arachidonic acidmetabolism or side effects to treatment with drugs acting on arachidonicacid metabolism.

[0484] Suitable examples of association studies using biallelic markersincluding the biallelic markers of the present invention, are studiesinvolving the following populations:

[0485] a case population suffering from a disease involving arachidonicacid metabolism and a healthy unaffected control population, or

[0486] a case population treated with agents acting on arachidonic acidmetabolism suffering from side-effects resulting from the treatment anda control population treated with the same agents showing noside-effects, or

[0487] a case population treated with agents acting on arachidonic acidmetabolism showing a beneficial response and a control populationtreated with same agents showing no beneficial response.

[0488] In a preferred embodiment, eicosanoid related-markers may be usedto identify individuals who are prone to hepatoxicity as a result ofdrug treatment. This includes diagnostic and prognostic assays toidentify individuals who are prone to liver toxicity as a result of drugtreatment, as well as clinical trials and treatment regimes whichutilize these assays. Said drug treatment may include any pharmaceuticalcompound suspected or known in the art to result in an increased levelof hepatoxicity.

[0489] In another preferred embodiment, the trait considered was a sideeffect upon drug treatment; the study involved two populations derivedfrom a clinical study of the anti-asthmatic drug zileuton. The casepopulation was composed of asthmatic individuals treated with Zileutonshowing zileuton-associated hepatotoxicity monitored by the serum levelof alanine aminotransferase (ALT) and the control population wascomposed of asthmatic individuals treated with zileuton and having noincreased serum level of ALT. Inclusion criteria and association betweenthe biallelic markers of the present invention and zileuton-associatedhepatotoxicity are further described below in IV.E. Association ofBiallelic Markers of the Invention with Hepatoxicity to Anti-Asthma DrugZileuton and in Example 5, Association between Side Effects uponTreatment with the Anti-Asthmatic Drug Zileuton (Zyflo™) and theBiallelic Markers of the 12-lipoxygenase Gene.

[0490] Association Analysis

[0491] The general strategy to perform association studies usingbiallelic markers derived from a region carrying a candidate gene is toscan two groups of individuals (case-control populations) in order tomeasure and statistically compare the allele frequencies of thebiallelic markers of the present invention in both groups.

[0492] If a statistically significant association with a trait isidentified for at least one or more of the analysed biallelic markers,one can assume that: either the associated allele is directlyresponsible for causing the trait (the associated allele is the traitcausing allele), or more likely the associated allele is in linkagedisequilibrium with the trait causing allele. The specificcharacteristics of the associated allele with respect to the candidategene function usually gives further insight into the relationshipbetween the associated allele and the trait (causal or in linkagedisequilibrium). If the evidence indicates that the associated allelewithin the candidate gene is most probably not the trait causing allelebut is in linkage disequilibrium with the real trait causing allele,then the trait causing allele can be found by sequencing the vicinity ofthe associated marker.

[0493] Association studies are usually run in two successive steps. In afirst phase, the frequencies of a reduced number of biallelic markersfrom one or several candidate genes are determined in the affected andcontrol populations. In a second phase of the analysis, the identity ofthe candidate gene and the position of the genetic loci responsible forthe given trait is further refined using a higher density of markersfrom the relevant region. However, if the candidate gene under study isrelatively small in length, as it is the case for many of the candidategenes analysed included in the present invention, a single phase may besufficient to establish significant associations.

[0494] Haplotype Analysis

[0495] As described above, when a chromosome carrying a disease allelefirst appears in a population as a result of either mutation ormigration, the mutant allele necessarily resides on a chromosome havinga set of linked markers: the ancestral haplotype. This haplotype can betracked through populations and its statistical association with a giventrait can be analysed. Complementing single point (allelic) associationstudies with multi-point association studies also called haplotypestudies increases the statistical power of association studies. Thus, ahaplotype association study allows one to define the frequency and thetype of the ancestral carrier haplotype. A haplotype analysis isimportant in that it increases the statistical power of an analysisinvolving individual markers.

[0496] In a first stage of a haplotype frequency analysis, the frequencyof the possible haplotypes based on various combinations of theidentified biallelic markers of the invention is determined. Thehaplotype frequency is then compared for distinct populations ofaffected and control individuals. The number of affected individuals,which should be, subjected to this analysis to obtain statisticallysignificant results usually ranges between 30 and 300, with a preferrednumber of individuals ranging between 50 and 150. The sameconsiderations apply to the number of unaffected individuals (or randomcontrol) used in the study. The results of this first analysis providehaplotype frequencies in case-control populations, for each evaluatedhaplotype frequency a p-value and an odd ratio are calculated. If astatistically significant association is found the relative risk for anindividual carrying the given haplotype of being affected with the traitunder study can be approximated.

[0497] Interaction Analysis

[0498] The biallelic markers of the present invention may also be usedto identify patterns of biallelic markers associated with detectabletraits resulting from polygenic interactions. The analysis of geneticinteraction between alleles at unlinked loci requires individualgenotyping using the techniques described herein. The analysis ofallelic interaction among a selected set of biallelic markers withappropriate level of statistical significance can be considered as ahaplotype analysis. Interaction analysis consists in stratifying thecase-control populations with respect to a given haplotype for the firstloci and performing a haplotype analysis with the second loci with eachsubpopulation.

[0499] Statistical methods used in association studies are furtherdescribed below in IV.C “Statistical Methods.”

[0500] 4. Testing for Linkage in the Presence of Association.

[0501] The biallelic markers of the present invention may further beused in TDT (transmission/disequilibrium test). TDT tests for bothlinkage and association and is not affected by populationstratification. TDT requires data for affected individuals and theirparents or data from unaffected sibs instead of from parents (seeSpielmann S. et al., Am. J. Hum. Genet., 52:506-516, 1993; Schaid D. J.et al., Genet. Epidemiol.,13:423-450, 1996, Spielmann S. and Ewens W.J., Am. J Hum. Genet., 62:450458, 1998). Such combined tests generallyreduce the false—positive errors produced by separate analyses.

[0502] C. Statistical Methods

[0503] In general, any method known in the art to test whether a traitand a genotype show a statistically significant correlation may be used.

[0504] 1. Methods in Linkage Analysis.

[0505] Statistical methods and computer programs useful for linkageanalysis are well-known to those skilled in the art (see Terwilliger J.D. and Ott J., Handbook of Human Genetic Linkage, John HopkinsUniversity Press, London, 1994; Ott J., Analysis ofHuman GeneticLinkage, John Hopkins University Press, Baltimore, 1991).

[0506] 2. Methods to Estimate Haplotype Frequencies in a Population.

[0507] As described above, when genotypes are scored, it is often notpossible to distinguish heterozygotes so that haplotype frequenciescannot be easily inferred. When the gametic phase is not known,haplotype frequencies can be estimated from the multilocus genotypicdata. Any method known to person skilled in the art can be used toestimate haplotype frequencies (see Lange K., Mathematical andStatistical Methods for Genetic Analysis, Springer, New York, 1997;Weir, B. S., Genetic data Analysis II: Methods for Discrete populationgenetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996)Preferably, maximum-likelihood haplotype frequencies are computed usingan Expectation- Maximization (EM) algorithm (see Dempster et al., J. R.Stat. Soc., 39B:1-38, 1977; Excoffier L. and Slatkin M., Mol. Biol.Evol., 12(5): 921-927, 1995). This procedure is an iterative processaiming at obtaining maximum-likelihood estimates of haplotypefrequencies from multi-locus genotype data when the gametic phase isunknown. Haplotype estimations are usually performed by applying the EMalgorithm using for example the EM-HAPLO program (Hawley M. E. et al.,Am. J. Phys. Anthropol., 18:104, 1994) or the Arlequin program(Schneider et al., Arlequin: a software for population genetics dataanalysis, University of Geneva, 1997). The EM algorithm is a generalisediterative maximum likelihood approach to estimation and is brieflydescribed below.

[0508] In what follows, phenotypes will refer to multi-locus genotypeswith unknown haplotypic phase. Genotypes will refer to mutli-locusgenotypes with known haplotypic phase.

[0509] Suppose one has a sample of N unrelated individuals typed for Kmarkers. The data observed are the unknown-phase K-locus phenotypes thatcan be categorized with F different phenotypes. Further, suppose that wehave H possible haplotypes (in the case of K biallelic markers, we havefor the maximum number of possible haplotypes H=2^(K)).

[0510] For phenotypej with c_(j) possible genotypes, we have:$\begin{matrix}{P_{j} = {{\sum\limits_{i = 1}^{c_{j}}{P\left( {{genotype}(i)} \right)}} = {\sum\limits_{i = 1}^{c_{j}}{{P\left( {h_{k},h_{l}} \right)}.}}}} & \text{Equation~~1}\end{matrix}$

[0511] Here, P_(j) is the probability of the j^(th) phenotype, andP(h_(k),h_(l)) is the probability of the i^(th) genotype composed ofhaplotypes h_(k) and h_(l). Under random mating (i.e. Hardy-WeinbergEquilibrium), P(h_(k)h_(l)) is expressed as:

P(h _(k) ,h _(l))=P(h _(k))² for h _(k) =h _(l), and

P(h _(k) ,h _(l))=2P(h _(k))P(h _(l)) for h _(k) ≠h _(l).   Equation 2

[0512] The E-M algorithm is composed of the following steps: First, thegenotype frequencies are estimated from a set of initial values ofhaplotype frequencies. These haplotype frequencies are denoted P₁ ⁽⁰⁾,P₂ ⁽⁰⁾, P₃ ⁽⁰⁾, . . . , P_(H) ⁽⁰⁾. The initial values for the haplotypefrequencies may be obtained from a random number generator or in someother way well known in the art. This step is referred to theExpectation step. The next step in the method, called the Maximizationstep, consists of using the estimates for the genotype frequencies tore-calculate the haplotype frequencies. The first iteration haplotypefrequency estimates are denoted by P₁ ⁽¹⁾, P₂ ⁽¹⁾, P₃ ⁽¹⁾, . . . , P_(H)⁽¹⁾. In general, the Expectation step at the s^(th) iteration consistsof calculating the probability of placing each phenotype into thedifferent possible genotypes based on the haplotype frequencies of theprevious iteration: $\begin{matrix}{{{P\left( {h_{k},h_{l}} \right)}^{(s)} = {\frac{n_{j}}{N}\left\lbrack \frac{{P_{j}\left( {h_{k},h_{l}} \right)}^{(s)}}{P_{j}} \right\rbrack}},} & \text{Equation~~3}\end{matrix}$

[0513] where n_(j) is the number of individuals with the j^(th)phenotype and P_(j)(h_(k), h_(l))^((s)) is the probability of genotypeh_(k),h_(l) in phenotype j. In the Maximization step, which isequivalent to the gene-counting method (Smith, Ann. Hum. Genet.,21:254-276, 1957), the haplotype frequencies are re-estimated based onthe genotype estimates: $\begin{matrix}{P_{t}^{({s + 1})} = {\frac{1}{2}{\sum\limits_{j = 1}^{F}{\sum\limits_{i = 1}^{c_{j}}{\delta_{it}{{P_{j}\left( {h_{k},h_{l}} \right)}^{(s)}.}}}}}} & \text{Equation~~4}\end{matrix}$

[0514] Here, δ_(it) is an indicator variable which counts the number ofoccurrences that haplotype t is present in i^(th) genotype; it takes onvalues 0, 1, and 2.

[0515] The E-M iterations cease when the following criterion has beenreached. Using Maximum Likelihood Estimation (MLE) theory, one assumesthat the phenotypes j are distributed multinomially. At each iterations, one can compute the likelihood function L. Convergence is achievedwhen the difference of the log-likehood between two consecutiveiterations is less than some small number, preferably 10⁻⁷.

[0516] 3. Methods to Calculate Linkage Disequilibrium Between Markers.

[0517] A number of methods can be used to calculate linkagedisequilibrium between any two genetic positions, in practice linkagedisequilibrium is measured by applying a statistical association test tohaplotype data taken from a population.

[0518] Linkage disequilibrium between any pair of biallelic markerscomprising at least one of the biallelic markers of the presentinvention (M_(i), M_(j)) having alleles (a_(i)/b_(i)) at marker M_(i)and alleles (a_(j)/b_(j)) at marker M_(j) can be calculated for everyallele combination (a_(i),a_(j); a_(i),b_(j); b_(i),a_(j) andb_(i),b_(j)), according to the Piazza formula:

Δ_(aiaj)={square root}θ4−{square root}(θ4+θ3) (θ4+θ2), where:

[0519] θ4=−−=frequency of genotypes not having allele a_(i) at M_(i) andnot having allele a_(j) at M_(j)

[0520] θ3=−+=frequency of genotypes not having allele a_(i) at M_(i) andhaving allele a_(j) at M_(j)

[0521] θ2=+−=frequency of genotypes having allele a_(i) at M_(i) and nothaving allele a_(j) at M_(j)

[0522] Linkage disequilibrium (LD) between pairs of biallelic markers(M_(i), M_(j)) can also be allocated for every allele combination(ai,aj; ai,bj; b_(i),a_(j) and b_(i),b_(j)), according to themaximum-likelihood estimate (MLE) for delta (the composite genotypicdisequilibrium coefficient), as described by Weir (Weir B. S., GeneticData Analysis, Sinauer Ass. Eds, 1996). The MLE for the compositelinkage disequilibrium is:

D _(aiaj)=(2n ₁ +n ₂ +n ₃ +n ₄/2)/N−2(pr(a _(i))·pr(a _(j)))

[0523] Where n₁=Σ phenotype (a_(i)/a_(i), a_(j)/a_(j)), n₂=Σ phenotype(a_(i)/a_(i), a_(j)), n₃=Σ phenotype (a_(i)/b_(i), a_(j)/a_(j)), n4=Σphenotype (a_(i)/b_(i), a_(j)/b_(j)) and N is the number of individualsin the sample.

[0524] This formula allows linkage disequilibrium between alleles to beestimated when only genotype, and not haplotype, data are available.

[0525] Another means of calculating the linkage disequilibrium betweenmarkers is as follows. For a couple of biallelic markers, M_(i)(a_(i)/b_(j)) and M_(j) (a_(j)/b_(j)), fitting the Hardy-Weinbergequilibrium, one can estimate the four possible haplotype frequencies ina given population according to the approach described above.

[0526] The estimation of gametic disequilibrium between ai and aj issimply:

D _(aiaj) =pr(haplotype(a _(i) ,a _(j)))−pr(a _(i))·pr(a _(j)).

[0527] Where pr(a_(i)) is the probability of allele a_(i) and pr(a_(j))is the probability of allele a_(j) and where pr(haplotype (a_(i),a_(j))) is estimated as in Equation 3 above.

[0528] For a couple of biallelic marker only one measure ofdisequilibrium is necessary to describe the association between M_(i)and M_(j).

[0529] Then a normalised value of the above is calculated as follows:

D′ _(aiaj) =D _(aiaj)/max(−pr(a _(i))·pr(a _(j)),−pr(b _(i))·pr(b _(j)))with D _(aiaj)<0

D′ _(aiaj) =D _(aiaj)/max(−pr(b _(i))·pr(a _(j)),−pr(a _(i))·pr(b _(j)))with D _(aiaj)<0

[0530] The skilled person will readily appreciate that other LDcalculation methods can be used without undue experimentation.

[0531] Linkage disequilibrium among a set of biallelic markers having anadequate heterozygosity rate can be determined by genotyping between 50and 1000 unrelated individuals, preferably between 75 and 200, morepreferably around 100.

[0532] 4. Testing for Association.

[0533] Methods for determining the statistical significance of acorrelation between a phenotype and a genotype, in this case an alleleat a biallelic marker or a haplotype made up of such alleles, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

[0534] Testing for association is performed by determining the frequencyof a biallelic marker allele in case and control populations andcomparing these frequencies with a statistical test to determine iftheir is a statistically significant difference in frequency which wouldindicate a correlation between the trait and the biallelic marker alleleunder study. Similarly, a haplotype analysis is performed by estimatingthe frequencies of all possible haplotypes for a given set of biallelicmarkers in case and control populations, and comparing these frequencieswith a statistical test to determine if their is a statisticallysignificant correlation between the haplotype and the phenotype (trait)under study. Any statistical tool useful to test for a statisticallysignificant association between a genotype and a phenotype may be used.Preferably the statistical test employed is a chi-square test with onedegree of freedom. A P-value is calculated (the P-value is theprobability that a statistic as large or larger than the observed onewould occur by chance).

[0535] Statistical Significance

[0536] In preferred embodiments, significance for diagnosis purposes,either as a positive basis for further diagnostic tests or as apreliminary starting point for early preventive therapy, the p valuerelated to a biallelic marker association is preferably about 1×10−2 orless, more preferably about 1 ×10−4 or less, for a single biallelicmarker analysis and about 1×10−3 or less, still more preferably 1×10−6or less and most preferably of about 1×10−8 or less, for a haplotypeanalysis involving several markers. These values are believed to beapplicable to any association studies involving single or multiplemarker combinations.

[0537] The skilled person can use the range of values set forth above asa starting point in order to carry out association studies withbiallelic markers of the present invention. In doing so, significantassociations between the biallelic markers of the present invention anddiseases involving arachidonic acid metabolism can be revealed and usedfor diagnosis and drug screening purposes.

[0538] Phenotypic Permutation

[0539] In order to confirm the statistical significance of the firststage haplotype analysis described above, it might be suitable toperform further analyses in which genotyping data from case-controlindividuals are pooled and randomised with respect to the traitphenotype. Each individual genotyping data is randomly allocated to twogroups, which contain the same number of individuals as the case-controlpopulations used to compile the data obtained in the first stage. Asecond stage haplotype analysis is preferably run on these artificialgroups, preferably for the markers included in the haplotype of thefirst stage analysis showing the highest relative risk coefficient. Thisexperiment is reiterated preferably at least between 100 and 10000times. The repeated iterations allow the determination of the percentageof obtained haplotypes with a significant p-value level.

[0540] Assessment of Statistical Association

[0541] To address the problem of false positives similar analysis may beperformed with the same case-control populations in random genomicregions. Results in random regions and the candidate region are comparedas described in U.S. Provisional Patent Application entitled “Methods,software and apparati for identifying genomic regions harbouring a geneassociated with a detectable trait”.

[0542] 5. Evaluation of Risk Factors.

[0543] The association between a risk factor (in genetic epidemiologythe risk factor is the presence or the absence of a certain allele orhaplotype at marker loci) and a disease is measured by the odds ratio(OR) and by the relative risk (RR). If P(R⁺) is the probability ofdeveloping the disease for individuals with R and P(R⁻) is theprobability for individuals without the risk factor, then the relativerisk is simply the ratio of the two probabilities, that is:

RR=P(R ⁺)/P(R ⁻)

[0544] In case-control studies, direct measures of the relative riskcannot be obtained because of the sampling design. However, the oddsratio allows a good approximation of the relative risk for low-incidencediseases and can be calculated:${OR} = {\left\lbrack \frac{F^{+}}{1 - F^{+}} \right\rbrack/\left\lbrack \frac{F^{-}}{\left( {1 - F^{-}} \right)} \right\rbrack}$

[0545] F⁺ is the frequency of the exposure to the risk factor in casesandF^(− is the frequency of the exposure to the risk factor in controls. F)⁺ and F⁻ are calculated using the allelic or haplotype frequencies ofthe study and further depend on the underlying genetic model (dominant,recessive, additive . . . ).

[0546] One can further estimate the attributable risk (AR) whichdescribes the proportion of individuals in a population exhibiting atrait due to a given risk factor. This measure is important inquantitating the role of a specific factor in disease etiology and interms of the public health impact of a risk factor. The public healthrelevance of this measure lies in estimating the proportion of cases ofdisease in the population that could be prevented if the exposure ofinterest were absent. AR is determined as follows:

AR=P _(E)(RR−1)/(P _(E)(RR−1)+1)

[0547] AR is the risk attributable to a biallelic marker allele or abiallelic marker haplotype. P_(E) is the frequency of exposure to anallele or a haplotype within the population at large; and RR is therelative risk which, is approximated with the odds ratio when the traitunder study has a relatively low incidence in the general population.

[0548] D. Association of Biallelic Markers of the Invention with Asthma

[0549] In the context of the present invention, an association betweenbiallelic marker alleles from candidate genes of the present inventionand a disease linked to arachidonic acid metabolism was demonstrated.The considered trait was asthma.

[0550] Asthma affects over 5% of the population in industrializedcountries. It is increasing in prevalence and severity and has a risingmortality (Rang H. P., Ritter J. M. and Dale M. M.; Pharmacology;Churchill Livingstone, NY, 1995). Bronchial asthma is a multifactorialsyndrome rather than a single disease, defined as airway obstructioncharacterized by inflammatory changes in the airways and bronchialhyper-responsiveness. In addition to the evidenced impact ofenvironmental factors on the development of asthma, patterns ofclustering and segregation in asthmatic families have suggested agenetic component to asthma. However the lack of a defined and specificasthma phenotype and of suitable markers for genetic analysis is provingto be a major hurdle for reliably identifying genes associated withasthma. The identification of genes implicated in asthma would representa major step towards the identification of new molecular targets for thedevelopment of anti-asthma drugs. Moreover there is no straightforwardphysiological or biological blood test for the asthmatic state. As aresult, adequate asthma treatment is often delayed, thereby allowing theinflammation process to better establish itself. Thus, there is a needfor the identification of asthma susceptibility genes in order todevelop an efficient and reliable asthma diagnostic test.

[0551] As mentioned above, products of arachidonic acid metabolism areimportant inflammatory mediators and have been involved in a number ofinflammatory diseases including asthma. More specifically,prostaglandins and leukotrienes are thought to play a major role in theinflammatory process observed in asthma patients.

[0552] In order to investigate and identify a genetic origin to asthma acandidate gene scan for asthma was conducted. The rational of thisapproach was to: 1) select candidate genes potentially involved in thepathological pathway of interest, in this case arachidonic acidmetabolism, 2) to identify biallelic markers in those genes and finally3) to measure the frequency of biallelic marker alleles in order todetermine if some alleles are more frequent in asthmatic populationsthan in non- affected populations. Results were further validated byhaplotype studies. Significant associations between biallelic markeralleles from the FLAP and 12-LO genes and asthma were demonstrated inthe context of the present invention. Association studies are furtherdescribed in Examples 3 and 4.

[0553] This information is extremely valuable. The knowledge of apotential genetic predisposition, even if this predisposition is notabsolute, might contribute in a very significant manner to treatmentefficacy of asthma patients and to the development of diagnostic tools.

[0554] E. Association of Biallelic Markers of the Invention withHepatotoxicity to Anti-Asthma Drug Zileuton (Zyflo™)

[0555] In the context of the present invention, an association betweenthe 12-LO gene and side effects related to treatment with theanti-asthmatic drug zileuton was discovered.

[0556] As mentioned above, bronchial asthma is a multifactorial syndromerather than a single disease, defined as airway obstructioncharacterized by inflammatory changes in the airways and bronchialhyper-responsiveness. Although initially reversible withbronchiodilators, airway obstruction becomes increasingly irreversibleif treated poorly. Asthma management therefore relies on early andregular use of drugs that control the disease. As a consequence, thereis a strong need for efficient and safe therapeutic opportunities forpatients with asthma. There are two main categories of anti-asthmaticdrugs—bronchodilators and anti-inflammatory agents. There is now generalagreement on the need to implement early anti-inflammatory treatmentrather than relying on symptomatic treatment with bronchiodilatorsalone. The leukotrienes, a family of proinflammatory mediators arisingvia arachidonic acid metabolism, have been implicated in theinflammatory cascade that occurs in asthmatic airways. Of greatrelevance to the pathogenesis of asthma is the 5-lipoxygenase, whichcatalyzes the initial step in the biosynthesis of leukotrienes fromarachidonic acid. Given the significant role of the inflammatory processin asthma, pharmacological agents, such as leukotriene antagonists and5-lipoxygenase inhibitors have been developed.

[0557] Zileuton (Zyflo™) is an active inhibitor of 5-lipoxygenase, theenzyme that catalyzes the formation of leukotrienes from arachidonicacid, indicated for prophylaxis and chronic treatment of asthma. Aminority of zileuton-treated patients develop liver functionabnormalities. Close monitoring revealed that elevations of liverfunction tests may occur during treatment with zileuton. The ALT test(serum level of alanine aminotransferase) was used, which is consideredthe most sensitive indicator of liver injury.

[0558] In order to investigate and identify a genetic origin tozileuton-associated hepatotoxicity, a candidate gene scan was conducted.This approach comprised:

[0559] selecting candidate genes potentially involved in thepathological pathway of interest or in the metabolism of zileuton, and

[0560] identifying biallelic markers in those genes, and finally

[0561] conducting association studies to identify biallelic markeralleles or haplotypes associated with elevations of liver function testsupon treatment with zileuton.

[0562] An association between elevated ALT levels upon treatment withzileuton and biallelic marker alleles from the 12-LO gene wasdemonstrated. Further details concerning this association study areprovided in Example 5.

[0563] F. Identification of Biallelic Markers in Linkage Disequilibriumwith the Biallelic Markers of the Invention

[0564] Once a first biallelic marker has been identified in a genomicregion of interest, the practitioner of ordinary skill in the art, usingthe teachings of the present invention, can easily identify additionalbiallelic markers in linkage disequilibrium with this first marker. Asmentioned before any marker in linkage disequilibrium with a firstmarker associated with a trait will be associated with the trait.Therefore, once an association has been demonstrated between a givenbiallelic marker and a trait, the discovery of additional biallelicmarkers associated with this trait is of great interest in order toincrease the density of biallelic markers in this particular region. Thecausal gene or mutation will be found in the vicinity of the marker orset of markers showing the highest correlation with the trait.

[0565] Identification of additional markers in linkage disequilibriumwith a given marker involves: (a) amplifying a genomic fragmentcomprising a first biallelic marker from a plurality of individuals; (b)identifying of second biallelic markers in the genomic region harboringsaid first biallelic marker; (c) conducting a linkage disequilibriumanalysis between said first biallelic marker and second biallelicmarkers; and (d) selecting said second biallelic markers as being inlinkage disequilibrium with said first marker. Subcombinationscomprising steps (b) and (c) are also contemplated.

[0566] Methods to identify biallelic markers and to conduct linkagedisequilibrium analysis are described herein and can be carried out bythe skilled person without undue experimentation. The present inventionthen also concerns biallelic markers which are in linkage disequilibriumwith the specific biallelic markers shown in Table 7(A-B) and which areexpected to present similar characteristics in terms of their respectiveassociation with a given trait.

[0567] G. Identification of Functional Mutations

[0568] Once a positive association is confirmed with a biallelic markerof the present invention, the associated candidate gene can be scannedfor mutations by comparing the sequences of a selected number ofaffected individuals and control individuals. In a preferred embodiment,functional regions such as exons and splice sites, promoters and otherregulatory regions of the candidate gene are scanned for mutations.Preferably, affected individuals carry the haplotype shown to beassociated with the trait and trait negative or control individuals donot carry the haplotype or allele associated with the trait. Themutation detection procedure is essentially similar to that used forbiallelic site identification.

[0569] The method used to detect such mutations generally comprises thefollowing steps: (a) amplification of a region of the candidate genecomprising a biallelic marker or a group of biallelic markers associatedwith the trait from DNA samples of affected patients and trait negativecontrols;

[0570] (b) sequencing of the amplified region;

[0571] (c) comparison of DNA sequences from affected trait-positivepatients and trait-negative controls; and

[0572] (d) determination of mutations specific to affectedtrait-positive patients. Subcombinations which comprise steps (b) and(c) are specifically contemplated.

[0573] It is preferred that candidate polymorphisms be then verified byscreening a larger population of cases and controls by means of anygenotyping procedure such as those described herein, preferably using amicrosequencing technique in an individual test format. Polymorphismsare considered as candidate mutations when present in cases and controlsat frequencies compatible with the expected association results.

[0574] Identification of mutations and low frequency polymorphisms inthe 5′ flanking region of the 12-LO gene, in the exons and introns ofthe 12-LO gene and in the 3′ flanking region of the 12-LO gene isfurther described in Example 5. Forty-nine low frequency polymorphismsand mutations were identified in the region of the 12-LO gene that wasscanned. Low frequency polymorphisms and mutations identified in exons5, 6, 8, and 13 are associated with amino acid substitutions at thepolypeptide level. In each of these amino acid substitutions theoriginal residue is replaced by a non-equivalent amino acid presentingdifferent chemical properties. As a consequence, specificity, activityand function of the 12-LO enzyme are modified. Biallelic marker10-343-231 is associated with a frame shift in the open reading frame ofthe 12-LO gene leading to the expression of a variant 12-LO polypeptidecomprising only 131 amino acids. This mutant 12-LO enzyme is probablyinactive or shows differences in specificity, activity and function.Biallelic marker 10-343-231 is associated with the deletion of a Leuresidue in the 12-LO polypeptide.

[0575] Candidate polymorphisms and mutations of the 12-LO gene suspectedof being responsible for the detectable phenotype, such as hepatoxicityto zileuton or asthma, can be confirmed by screening a larger populationof affected and unaffected individuals using any of the genotypingprocedures described herein. Preferably the microsequencing technique isused. Such polymorphisms are considered as candidate “trait-causing”mutations when they exhibit a statistically significant correlation withthe detectable phenotype.

[0576] V. Biallelic Markers of the Invention in Methods of GeneticDiagnostics

[0577] The biallelic markers of the present invention can also be usedto develop diagnostics tests capable of identifying individuals whoexpress a detectable trait as the result of a specific genotype orindividuals whose genotype places them at risk of developing adetectable trait at a subsequent time. The trait analyzed using thepresent diagnostics may be any detectable trait, including a diseaseinvolving arachidonic acid metabolism, a response to an agent acting onarachidonic acid metabolism or side effects to an agent acting onarachidonic acid metabolism.

[0578] The diagnostic techniques of the present invention may employ avariety of methodologies to determine whether a test subject has abiallelic marker pattern associated with an increased risk of developinga detectable trait or whether the individual suffers from a detectabletrait as a result of a particular mutation, including methods whichenable the analysis of individual chromosomes for haplotyping, such asfamily studies, single sperm DNA analysis or somatic hybrids.

[0579] The present invention provides diagnostic methods to determinewhether an individual is at risk of developing a disease or suffers froma disease resulting from a mutation or a polymorphism in a candidategene of the present invention. The present invention also providesmethods to determine whether an individual is likely to respondpositively to an agent acting on arachidonic acid metabolism or whetheran individual is at risk of developing an adverse side effect to anagent acting on arachidonic acid metabolism.

[0580] These methods involve obtaining a nucleic acid sample from theindividual and, determining, whether the nucleic acid sample contains atleast one allele or at least one biallelic marker haplotype, indicativeof a risk of developing the trait or indicative that the individualexpresses the trait as a result of possessing a particular candidategene polymorphism or mutation (trait-causing allele).

[0581] Preferably, in such diagnostic methods, a nucleic acid sample isobtained from the individual and this sample is genotyped using methodsdescribed above in III. Methods of Genotyping an Individual forBiallelic Markers. The diagnostics may be based on a single biallelicmarker or a on group of biallelic markers.

[0582] In each of these methods, a nucleic acid sample is obtained fromthe test subject and the biallelic marker pattern of one or more of thebiallelic markers listed in Table 7(A-B) is determined.

[0583] In one embodiment, a PCR amplification is conducted on thenucleic acid sample to amplify regions in which polymorphisms associatedwith a detectable phenotype have been identified. The amplificationproducts are sequenced to determine whether the individual possesses oneor more polymorphisms associated with a detectable phenotype. Theprimers used to generate amplification products may comprise the primerslisted in Table 13, or a preferred set of primers includes thosedescribed in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539,541-646, and 651-652. Alternatively, the nucleic acid sample issubjected to microsequencing reactions as described above to determinewhether the individual possesses one or more polymorphisms associatedwith a detectable phenotype resulting from a mutation or a polymorphismin a candidate gene. The primers used in the microsequencing reactionsmay include the primers listed in Table 12, or a preferred set ofprimers includes those described in SEQ ID Nos. 26-70, 72418,425-489,491-530, 532-539, 541-646, and 651-652. In another embodiment,the nucleic acid sample is contacted with one or more allele specificoligonucleotide probes which, specifically hybridize to one or morecandidate gene alleles associated with a detectable phenotype. Theprobes used in the hybridization assay may include the probes listed inTable 14, or a preferred set of probes includes those described in SEQID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.

[0584] The present invention provides methods of determining whether anindividual is at risk of developing asthma, or whether said individualsuffers from asthma, comprising: a) genotyping said individual for atleast one 12-LO-related biallelic marker; and b) correlating the resultof step a) with a risk of developing asthma. In a preferred embodiment,said 12-LO-related biallelic marker is selected from the groupconsisting of biallelic markers: 12-197-244, 12-208-35, 12-226-167,12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220,10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230,and 12-223-207. Preferably, said 12-LO-related biallelic marker isselected from the biallelic markers described in Example 5. The presentinvention also provides methods of determining whether an individual isat risk of developing hepatoxicity upon treatment with zileuton,comprising: a) genotyping said individual for at least one 12-LO-relatedbiallelic marker; and b) correlating the result of step a) with a riskof developing hepatotoxicity upon treatment with zileuton. In apreferred embodiment, said 12-LO-related biallelic marker is selectedfrom the group consisting of biallelic markers : 12-197-244, 12-208-35,12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-220,10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421,12-219-230, and 12-223- 207. Preferably, said 12-LO-related biallelicmarker is selected from the biallelic markers described in Example 5,Association between Side Effects upon Treatment with the Anti-AsthmaticDrug Zileuton (Zyflo™) and the Biallelic Markers of the 12-lipoxygenaseGene.

[0585] These diagnostic methods are extremely valuable as they can, incertain circumstances, be used to initiate preventive treatments or toallow an individual carrying a significant haplotype to foresee warningsigns such as minor symptoms. In diseases in which attacks may beextremely violent and sometimes fatal if not treated on time, such asasthma, the knowledge of a potential predisposition, even if thispredisposition is not absolute, might contribute in a very significantmanner to treatment efficacy. Similarly, a diagnosed predisposition to apotential side effect could immediately direct the physician toward atreatment for which such side effects have not been observed duringclinical trials.

[0586] Diagnostics, which analyze and predict response to a drug or sideeffects to a drug, may be used to determine whether an individual shouldbe treated with a particular drug. For example, if the diagnosticindicates a likelihood that an individual will respond positively totreatment with a particular drug, the drug may be administered to theindividual. Conversely, if the diagnostic indicates that an individualis likely to respond negatively to treatment with a particular drug, analternative course of treatment may be prescribed. A negative responsemay be defined as either the absence of an efficacious response or thepresence of toxic side effects.

[0587] Clinical drug trials represent another application for themarkers of the present invention. One or more markers indicative ofresponse to an agent acting on arachidonic acid metabolism or to sideeffects to an agent acting on arachidonic acid metabolism may beidentified using the methods described above. Thereafter, potentialparticipants in clinical trials of such an agent may be screened toidentify those individuals most likely to respond favorably to the drugand exclude those likely to experience side effects. In that way, theeffectiveness of drug treatment may be measured in individuals whorespond positively to the drug, without lowering the measurement as aresult of the inclusion of individuals who are unlikely to respondpositively in the study and without risking undesirable safety problems.

[0588] VI. Computer-Related Embodiments

[0589] As used herein the term “nucleic acid codes of the invention”encompass the nucleotide sequences comprising, consisting essentiallyof, or consisting of any one of the following: a) a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500 or 1000 nucleotides, to the extent that a polynucleotide ofthese lengths is consistent with the lengths of the particular SequenceID, of a sequence selected from the group consisting of the sequencesdescribed in Table 8, and the complements thereof, excluding Sequence IDNos. 1-10, 19, 23-25, and 647-650; b) a contiguous span of at least 12,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or1000 nucleotides, to the extent that a polynucleotide of these lengthsis consistent with the lengths of the particular Sequence ID, of asequence selected from the group consisting of the sequences describedin Table 9, and the complements thereof, excluding Sequence ID Nos.11-18 and 20-21; c) a contiguous span of at least 12, 15, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 500 nucleotides, tothe extent that a polynucleotide of these lengths is consistent with thelengths of the particular Sequence ID, of a sequence selected from thegroup consisting of the sequences described in Table 12, more preferablya set of markers or sequences consisting of those markers or sequencesfound in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646,and 651-652, and the complements thereof, wherein said span includes aneicosanoid-related biallelic marker, preferably an eicosanoid-relatedbiallelic marker described in Table 7(A-B), preferably the biallelicmarkers found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539,541-646, and 651-652, or more preferably from SEQ ID Nos. 651-652,680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, insaid sequence with the alternative allele present at said biallelicmarker.

[0590] The “nucleic acid codes of the invention” further encompassnucleotide sequences homologous to a contiguous span of at least 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to theextent that a contiguous span of these lengths is consistent with thelengths of the particular Sequence ID, of a sequence selected from thegroup consisting of the sequences described in Tables 8, 9, and 12, andthe complements thereof. Homologous sequences refer to a sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology tothese contiguous spans. Homology may be determined using any methoddescribed herein, including BLAST2N with the default parameters or withany modified parameters. Homologous sequences also may include RNAsequences in which uridines replace the thymines in the nucleic acidcodes of the invention. It will be appreciated that the nucleic acidcodes of the invention can be represented in the traditional singlecharacter format (See the inside back cover of Stryer, Lubert.Biochemistry, 3^(rd) edition. W. H Freeman & Co., New York.) or in anyother format or code which records the identity of the nucleotides in asequence.

[0591] It will be appreciated by those skilled in the art that thenucleic acid codes of the invention, one or more of the polypeptidecodes of SEQ ID Nos. 653 and 654 can be stored, recorded, andmanipulated on any medium which can be read and accessed by a computer.As used herein, the words “recorded” and “stored” refer to a process forstoring information on a computer medium. A skilled artisan can readilyadopt any of the presently known methods for recording information on acomputer readable medium to generate manufactures comprising one or moreof the nucleic acid codes of the invention and one or more of thepolypeptide codes of SEQ ID Nos. 653-654. Another aspect of the presentinvention is a computer readable medium having recorded thereon at least2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes of the invention, andthe complements thereof. Another aspect of the present invention is acomputer readable medium having recorded thereon at least 2, 5, 10, 15,20, 25, 30, or 50 polypeptide codes of SEQ ID Nos. 653-654.

[0592] Computer readable media include magnetically readable media,optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

[0593] Embodiments of the present invention include systems,particularly computer systems which store and manipulate the sequenceinformation described herein. As used herein, “a computer system” refersto the hardware components, software components, and data storagecomponents used to analyze the nucleotide sequences of the nucleic acidcodes of the invention , or the amino acid sequences of the polypeptidecodes of SEQ ID Nos. 653-654. In one embodiment, the computer system isa Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). Thecomputer system preferably includes a processor for processing,accessing and manipulating the sequence data. The processor can be anywell-known type of central processing unit, such as the Pentium III fromIntel Corporation, or similar processor from Sun, Motorola, Compaq orInternational Business Machines. Preferably, the computer system is ageneral purpose system that comprises the processor and one or moreinternal data storage components for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable. In one particularembodiment, the computer system includes a processor connected to a buswhich is connected to a main memory (preferably implemented as RAM) andone or more internal data storage devices, such as a hard drive and/orother computer readable media having data recorded thereon. In someembodiments, the computer system further includes one or more dataretrieving device for reading the data stored on the internal datastorage devices. The data retrieving device may represent, for example,a floppy disk drive, a compact disk drive, a magnetic tape drive, etc.In some embodiments, the internal data storage device is a removablecomputer readable medium such as a floppy disk, a compact disk, amagnetic tape, etc. containing control logic and/or data recordedthereon. The computer system may advantageously include or be programmedby appropriate software for reading the control logic and/or the datafrom the data storage component once inserted in the data retrievingdevice. The computer system includes a display which is used to displayoutput to a computer user. It should also be noted that the computersystem can be linked to other computer systems in a network or wide areanetwork to provide centralized access to the computer system. Softwarefor accessing and processing the nucleotide sequences of the nucleicacid codes of the invention, or the amino acid sequences of thepolypeptide codes of SEQ ID Nos. 653-654 (such as search tools, comparetools, and modeling tools etc.) may reside in main memory duringexecution. In some embodiments, the computer system may further comprisea sequence comparer for comparing the above-described nucleic acid codesof the invention or polypeptide codes of SEQ ID Nos. 653-654 stored on acomputer readable medium to reference nucleotide or polypeptidesequences stored on a computer readable medium. A “sequence comparer”refers to one or more programs which are implemented on the computersystem to compare a nucleotide or polypeptide sequence with othernucleotide or polypeptide sequences and/or compounds including but notlimited to peptides, peptidomimetics, and chemicals stored within thedata storage means. For example, the sequence comparer may compare thenucleotide sequences of the nucleic acid codes of the invention, or theamino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654stored on a computer readable medium to reference sequences stored on acomputer readable medium to identify homologies, motifs implicated inbiological function, or structural motifs. The various sequence comparerprograms identified elsewhere in this patent specification areparticularly contemplated for use in this aspect of the invention.

[0594] One embodiment is a process for comparing a new nucleotide orprotein sequence with a database of sequences in order to determine thehomology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system, or a public database such as GENBANK, PIR ORSWISSPROT that is available through the Internet.

[0595] The process begins at a start state and then moves to a statewherein the new sequence to be compared is stored to a memory in acomputer system. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device. The process thenmoves to a state wherein a database of sequences is opened for analysisand comparison. The process then moves to a state wherein the firstsequence stored in the database is read into a memory on the computer. Acomparison is then performed at a state to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are id introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate, a determination is made at a decision state whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess. If a determination is made that the two sequences are the same,the process moves to a state wherein the name of the sequence from thedatabase is displayed to the user. This state notifies the user that thesequence with the displayed name fulfills the homology constraints thatwere entered. Once the name of the stored sequence is displayed to theuser, the process moves to a decision state wherein a determination ismade whether more sequences exist in the database. If no more sequencesexist in the database, then the process terminates at an end state.However, if more sequences do exist in the database, then the processmoves to a state wherein a pointer is moved to the next sequence in thedatabase so that it can be compared to the new sequence. In this manner,the new sequence is aligned and compared with every sequence in thedatabase. It should be noted that if a determination had been made atthe decision statethat the sequences were not homologous, then theprocess would move imrnediately to the decision state in order todetermine if any other sequences were available in the database forcomparison. Accordingly, one aspect of the present invention is acomputer system comprising a processor, a data storage device havingstored thereon a nucleic acid code of the invention or a polypeptidecode of SEQ ID Nos. 653-654, a data storage device having retrievablystored thereon reference nucleotide sequences or polypeptide sequencesto be compared to the nucleic acid code of the invention or polypeptidecode of SEQ ID Nos. 653-654 and a sequence comparer for conducting thecomparison. The sequence comparer may indicate a homology level betweenthe sequences compared or identify structural motifs in the abovedescribed nucleic acid code of the invention and polypeptide codes ofSEQ ID Nos. 653-654 or it may identify structural motifs in sequenceswhich are compared to these nucleic acid codes and polypeptide codes. Insome embodiments, the data storage device may have stored thereon thesequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleicacid codes of the invention or polypeptide codes of SEQ ID Nos. 653-654.

[0596] Another aspect of the present invention is a method fordetermining the level of homology between a nucleic acid code of theinvention and a reference nucleotide sequence, comprising the steps ofreading the nucleic acid code and the reference nucleotide sequencethrough the use of a computer program which determines homology levelsand determining homology between the nucleic acid code and the referencenucleotide sequence with the computer program. The computer program maybe any of a number of computer programs for determining homology levels,including those specifically enumerated herein, including BLAST2N withthe default parameters or with any modified parameters. The method maybe implemented using the computer systems described above. The methodmay also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of theabove described nucleic acid codes of the invention through use of thecomputer program and determining homology between the nucleic acid codesand reference nucleotide sequences .

[0597] One embodiment is a process in a computer for determining whethertwo sequences are homologous. The process begins at a start state andthen moves to a state wherein a first sequence to be compared is storedto a memory. The second sequence to be compared is then stored to amemory at a state. The process then moves to a state wherein the firstcharacter in the first sequence is read and then to a state wherein thefirst character of the second sequence is read. It should be understoodthat if the sequence is a nucleotide sequence, then the character wouldnormally be either A, T, C, G or U. If the sequence is a proteinsequence, then it should be in the single letter amino acid code so thatthe first and sequence sequences can be easily compared.

[0598] A determination is then made at a decision state whether the twocharacters are the same. If they are the same, then the process moves toa state wherein the next characters in the first and second sequencesare read. A determination is then made whether the next characters arethe same. If they are, then the process continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process moves to a decision stateto determine whether there are any more characters either sequence toread. If there aren't any more characters to read, then the processmoves to a state wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%. Alternatively, the computer program may bea computer program which compares the nucleotide sequences of thenucleic acid codes of the present invention, to reference nucleotidesequences in order to determine whether the nucleic acid code of theinvention differs from a reference nucleic acid sequence at one or morepositions. Optionally such a program records the length and identity ofinserted, deleted or substituted nucleotides with respect to thesequence of either the reference polynucleotide or the nucleic acid codeof the invention. In one embodiment, the computer program may be aprogram which determines whether the nucleotide sequences of the nucleicacid codes of the invention contain a biallelic marker or singlenucleotide polymorphism (SNP) with respect to a reference nucleotidesequence. This single nucleotide polymorphism may comprise a single basesubstitution, insertion, or deletion, while this biallelic marker maycomprise about one to ten consecutive bases substituted, inserted ordeleted.

[0599] Another aspect of the present invention is a method fordetermining the level of homology between a polypeptide code of SEQ IDNos. 653-654 and a reference polypeptide sequence, comprising the stepsof reading the polypeptide code of SEQ ID Nos. 653-654 and the referencepolypeptide sequence through use of a computer program which determineshomology levels and determining homology between the polypeptide codeand the reference polypeptide sequence using the computer program.

[0600] Accordingly, another aspect of the present invention is a methodfor determining whether a nucleic acid code of the invention differs atone or more nucleotides from a reference nucleotide sequence comprisingthe steps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above. The method may alsobe performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of thenucleic acid codes of the invention and the reference nucleotidesequences through the use of the computer program and identifyingdifferences between the nucleic acid codes and the reference nucleotidesequences with the computer program. In other embodiments the computerbased system may further comprise an identifier for identifying featureswithin the nucleotide sequences of the nucleic acid codes of theinvention or the amino acid sequences of the polypeptide codes of SEQ IDNos. 653-654. An “identifier” refers to one or more programs whichidentifies certain features within the above-described nucleotidesequences of the nucleic acid codes of the invention or the amino acidsequences of the polypeptide codes of SEQ ID Nos. 653-654. In oneembodiment, the identifier may comprise a program which identifies anopen reading frame in the cDNAs codes of SEQ ID No. 652.

[0601] One embodiment is an identifier process for detecting thepresence of a feature in a sequence. The process begins at a start stateand then moves to a state wherein a first sequence that is to be checkedfor features is stored to a memory in the computer system. The processthen moves to a state wherein a database of sequence features is opened.Such a database would include a list of each feature's attributes alongwith the name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG.” Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group (www.gcg.com). Once the database offeatures is opened at the state, the process moves to a state whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate. A determination is then made at a decision state whether theattribute of the feature was found in the first sequence. If theattribute was found, then the process moves to a state 318 wherein thename of the found feature is displayed to the user. The process thenmoves to a decision state wherein a determination is made whether movefeatures exist in the database. If no more features do exist, then theprocess terminates at an end state. However, if more features do existin the database, then the process reads the next sequence feature at astate and loops back to the state wherein the attribute of the nextfeature is compared against the first sequence. It should be noted, thatif the feature attribute is not found in the first sequence at thedecision state, the process moves directly to the decision state inorder to determine if any more features exist in the database. Inanother embodiment, the identifier may comprise a molecular modelingprogram which determines the 3-dimensional structure of the polypeptidescodes of SEQ ID Nos. 653-654. In some embodiments, the molecularmodeling program identifies target sequences that are most compatiblewith profiles representing the structural environments of the residuesin known three-dimensional protein structures. (See, e.g., Eisenberg etal., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, the disclosure ofwhich is incorporated herein by reference in its entirety). In anothertechnique, the known three-dimensional structures of proteins in a givenfamily are superimposed to define the structurally conserved regions inthat family. This protein modeling technique also uses the knownthree-dimensional structure of a homologous protein to approximate thestructure of the polypeptide codes of SEQ ID Nos. 653-654. (See e.g.,Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, thedisclosure of which is incorporated herein by reference in itsentirety). Conventional homology modeling techniques have been usedroutinely to build models of proteases and antibodies. (Sowdhamini etal., Protein Engineering 10:207, 215 (1997), the disclosure of which isincorporated herein by reference in its entirety). Comparativeapproaches can also be used to develop three-dimensional protein modelswhen the protein of interest has poor sequence identity to templateproteins. In some cases, proteins fold into similar three-dimensionalstructures despite having very weak sequence identities. For example,the three-dimensional structures of a number of helical cytokines foldin similar three-dimensional topology in spite of weak sequencehomology. The recent development of threading methods now enables theidentification of likely folding patterns in a number of situationswhere the structural relatedness between target and template(s) is notdetectable at the sequence level. Hybrid methods, in which foldrecognition is performed using Multiple Sequence Threading (MST),structural equivalencies are deduced from the threading output using adistance geometry program DRAGON to construct a low resolution model,and a full-atom representation is constructed using a molecular modelingpackage such as QUANTA.

[0602] According to this 3-step approach, candidate templates are firstidentified by using the novel fold recognition algorithm MST, which iscapable of performing simultaneous threading of multiple alignedsequences onto one or more 3-D structures. In a second step, thestructural equivalencies obtained from the MST output are converted intointerresidue distance restraints and fed into the distance geometryprogram DRAGON, together with auxiliary information obtained fromsecondary structure predictions. The program combines the restraints inan unbiased manner and rapidly generates a large number of lowresolution model confirmations. In a third step, these low resolutionmodel confirmations are converted into full-atom models and subjected toenergy minimization using the molecular modeling package QUANTA. (Seee.g., Aszódi et al., Proteins: Structure, Function, and Genetics,Supplement 1:38-42 (1997), the disclosure of which is incorporatedherein by reference in its entirety).

[0603] The results of the molecular modeling analysis may then be usedin rational drug design techniques to identify agents which modulate theactivity of the polypeptide codes of SEQ ID Nos. 653-654. Accordingly,another aspect of the present invention is a method of identifying afeature within the nucleic acid codes of the invention or thepolypeptide codes of SEQ ID Nos. 653-654 comprising reading the nucleicacid code(s) or the polypeptide code(s) through the use of a computerprogram which identifies features therein and identifying featureswithin the nucleic acid code(s) or polypeptide code(s) with the computerprogram. In one embodiment, computer program comprises a computerprogram which identifies open reading frames. In a further embodiment,the computer program identifies structural motifs in a polypeptidesequence. In another embodiment, the computer program comprises amolecular modeling program. The method may be performed by reading asingle sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of thenucleic acid codes of the invention or the polypeptide codes of SEQ IDNos. 653-654 through the use of the computer program and identifyingfeatures within the nucleic acid codes or polypeptide codes with thecomputer program. The nucleic acid codes of the invention or thepolypeptide codes of SEQ ID Nos. 653-654 may be stored and manipulatedin a variety of data processor programs in a variety of formats. Forexample, the nucleic acid codes of the invention or the polypeptidecodes of SEQ ID Nos. 653-654 may be stored as text in a word processingfile, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in avariety of database programs familiar to those of skill in the art, suchas DB2, SYBASE, or ORACLE. In addition, many computer programs anddatabases may be used as sequence comparers, identifiers, or sources ofreference nucleotide or polypeptide sequences to be compared to thenucleic acid codes of the invention or the polypeptide codes of SEQ IDNos. 653-654. The following list is intended not to limit the inventionbut to provide guidance to programs and databases which are useful withthe nucleic acid codes of the invention or the polypeptide codes of SEQID No. 653-654. The programs and databases which may be used include,but are not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403 (1990), the disclosure of which is incorporated herein byreference in its entirety), FASTA (Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85: 2444 (1988), the disclosure of which is incorporatedherein by reference in its entirety), FASTDB (Brutlag et al. Comp. App.Biosci. 6:237-245, 1990, the disclosure of which is incorporated hereinby reference in its entirety), Catalyst (Molecular Simulations Inc.),Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (MolecularSimulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II,(Molecular Simulations Inc.), Discover molecular Simulations Inc.),CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the EMBL/Swissprotein database, the MDL Available ChemicalsDirectory database, the MDL Drug Data Report data base, theComprehensive Medicinal Chemistry database, Derwents's World Drug Indexdatabase, the BioByteMasterFile database, the Genbank database, and theGenseqn database. Many other programs and data bases would be apparentto one of skill in the art given the present disclosure. Motifs whichmay be detected using the above programs include sequences encodingleucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

[0604] It should be noted that the nucleic acid codes of the inventionfurther encompass all of the polynucleotides disclosed, described orclaimed in the present invention. Also, it should be noted that thepolypeptide codes of SEQ ID Nos. 653-654 further encompass all of thepolypeptides disclosed, described or claimed in the present invention.Moreover, the present invention specifically contemplates the storage ofsuch codes on computer readable media and computer systems individuallyor in combination, as well as the use of such codes and combinations inthe methods of section “VI. Computer-Related Embodiments.”

[0605] Throughout this application, various publications, patents, andpublished patent applications are cited. The disclosures of thepublications, patents, and published patent specifications referenced inthis application are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

[0606] VII. DNA Typing Methods and Systems

[0607] The present invention also encompasses a DNA typing system havinga much higher discriminatory power than currently available typingsystems. The systems and associated methods are particularly applicablein the identification of individuals for forensic science and paternitydeterminations. These applications have become increasingly important;in forensic science, for example, the identification of individuals bypolymorphism analysis has become widely accepted by courts as evidence.

[0608] While forensic geneticists have developed many techniques tocompare homologous segments of DNA to determine if the segments areidentical or if they differ in one or more nucleotides, each techniquestill has certain disadvantages. In particular, the techniques varywidely in terms of expense of analysis, time required to carry out ananalysis and statistical power.

[0609] RFLP Analysis Methods

[0610] The best known and most widespread method in forensic DNA typingis the restriction fragment length polymorphism (RFLP) analysis. In RFLPtesting, a repetitive DNA sequence referred to as a variable numbertandem repeat (VNTR) which varies between individuals is analyzed. Thecore repeat is typically a sequence of about 15 base pairs in length,and highly polymorphic VNTR loci can have an average of about 20alleles. DNA restriction sites located on either site of the VNTR areexploited to create DNA fragments from about 0.5 Kb to less than 10 Kbwhich are then separated by electrophoresis, indicating the number ofrepeats found in the individual at the particular loci. RFLP methodsgenerally consist of (1) extraction and isolation of DNA, (2)restriction endonuclease digestion; (3) separation of DNA fragments byelectrophoresis; (4) capillary transfer; (5) hybridization withradiolabelled probes; (6) autoradiography; and (7) interpretation ofresults (Lee, H. C. et al., Am. J. Forensic. Med. Pathol. 15(4): 269-282(1994)). RFLP methods generally combine analysis at about 5 loci andhave much higher discriminate potential than other available test duethe highly polymorphic nature of the VNTRs. However, autoradiography iscostly and time consuming and an analysis generally takes weeks ormonths for turnaround. Additionally, a large amount of sample DNA isrequired, which is often not available at a crime scene. Furthermore,the reliability of the system and its credibility as evidence isdecreased because the analysis of tightly spaced bands onelectrophoresis results in a high rate of error.

[0611] PCR Methods

[0612] PCR based methods offer an alternative to RFLP methods. In afirst method called AmpFLP, DNA fragments containing VNTRs are amplifiedand then separated electrophoretically, without the restriction step ofRFLP method. While this method allows small quantities of sample DNA tobe used, decreases analysis time by avoiding autoradiography, andretains high discriminatory potential, it nevertheless requireselectrophoretic separation which takes substantial time and introducesan significant error rate. In another AmpFLP method, short tandemrepeats (STRs) of 2 to 8 base pairs are analyzed. STRs are more suitableto analysis of degraded DNA samples since they require smaller amplifiedfragments but have the disadvantage of requiring separation of theamplified fragments. While STRs are far less informative than longerrepeats, similar discriminatory potential can be achieved if enough STRsare used in a single analysis.

[0613] Other methods include sequencing of mitochondrial DNA, which isespecially suitable for situations where sample DNA is very degraded orin small quantities. However, only a small region of 1 Kb of themitochondrial DNA referred to as the D-Loop locus has been found usefulfor typing because of its polymorphic nature, resulting in lowerdiscriminatory potential than with RFLP or AmpFLP methods. Furthermore,DNA sequencing is expensive to carry out on a large number of samples.

[0614] Further available methods include dot-blot methods, which involveusing allele specific oligonucleotide probes which hybridize sequencespecifically to one allele of a polymorphic site. Systems include theHLA DQ-alpha kit developed by Cetus Corp. which has a discriminatoryvalue of about 1 in 20, and a dot-blot strip referred to as thePolymarker strip combining five genetic loci for a discriminatory valueof about one in a few thousand. (Weedn, V., Clinics in Lab. Med. 16(1):187-196 (1996)).

[0615] In addition to difficulties in analysis and time consuminglaboratory procedures, it remains desirable for all DNA typing systemsto have a higher discriminatory power. Several applications exist inwhich even the most discriminating tests need improvement in order toremove the considerable remaining doubt resulting from such analyses.Table 3 below lists characteristics of currently available forensictesting systems (Weedn, (1996)) and compares them with the method of theinvention. TABLE 3 Sensitivity Turnaround Discriminatory (amount Testtype Technology time potential DNA) Sample RFLP VNTR Weeks or 10⁶ to 10⁹ 10 ng Highly intact (autoradiography) months DNA AmpFLP VNTR Days 10³to 10⁶ 100 pg Moderate (PCR based) degradation Dot blot (ex. Sequencespecific Days 10¹ to 10³  1 ng Moderate HLADQA1) oligonucleotidedegradation probes Mitochondrial D-loop sequence Days 10²  1 pg SevereDNA (PCR based) degradation Present marker Biallelic Markers Hours to10⁶, 10⁴⁷, 10⁶⁵⁰ 100 pg Moderate set of the (set of 13, set of Daysdegradation invention 100, set of 500, set (throughput of 650)dependent)

[0616] Applications

[0617] As described above, an important application of DNA typing testsis to determine whether a DNA sample (e.g. from a crime scene)originated from an individual suspected of leaving said DNA sample.

[0618] There are several applications for DNA typing which require aparticularly powerful genotyping system. In a first application, a highpowered typing system is advantageous when for example a suspect isidentified by searching a DNA profile database such as that maintainedby the U.S. Federal Bureau of Investigation. Since databases may containlarge numbers of data entries that are expected to increaseconsistently, currently used forensic systems can be expected toidentify several matching DNA profiles due to their relative lack ofpower. While database searches generally reinforce the evidence byexcluding other possible suspects, low powered typing systems resultingin the identification of several individuals may often tend to diminishthe overall case against a defendant.

[0619] In another application, a target population is systematicallytested to identify an individual having the same DNA profile as that ofa DNA sample. In such a situation, a defendant is chosen at random basedon DNA profile from a large population of innocent individuals. Sincethe population tested can often be large enough that at least onepositive match is identified, and it is usually not possible toexhaustively test a population, the usefulness of the evidence willdepend on the level of significance of the forensic test. In order torender such an application useful as a sole or primary source ofevidence, DNA typing systems of extremely high discriminatory potentialare required.

[0620] In yet another application, it is desirable to be able todiscriminate between related individuals. Because related individualswill be expected to share a large portion of alleles at polymorphicsites, a very high powered DNA typing assay would be required todiscriminate between them. This can have important effects if a sampleis found to match the defendant's DNA profile and no evidence that theperpetrator is a relative can be found.

[0621] Accordingly, there a need in this art for a rapid, simple,inexpensive and accurate technique having a very high resolution valueto determine relationships between individuals and differences in degreeof relationships. Also, there is a need in the art for a very accurategenetic relationship test procedure which uses very small amounts of anoriginal DNA sample, yet produces very accurate results.

[0622] The present invention thus involves methods for theidentification of individuals comprising determining the identity of thenucleotides at set of genetic markers in a biological sample, whereinsaid set of genetic markers comprises at least one eicosanoid-relatedbiallelic marker. The present invention provides an extensive set ofbiallelic markers allowing a higher discriminatory potential than thegenetic markers used in current forensic typing systems. Also, biallelicmarkers can be genotyped in individuals with much higher efficiency andaccuracy than the genetic markers used in current forensic typingsystems. In preferred embodiments, the invention comprises determiningthe identity of a nucleotide at an eicosanoid-related biallelic markerby single nucleotide primer extension, which does not requireelectrophoresis as in techniques described above and results in lowerrate of experimental error. As shown in Table 3, herein, in comparisonwith PCR based VNTR based methods which allow discriminatory potentialof thousands to millions, and RFLP based methods which allowdiscriminatory potential of merely millions to billions under optimalassumptions, the biallelic marker based method of the present inventionprovides a radical increase in discriminatory potential.

[0623] Any suitable set of genetic markers and biallelic markers of theinvention may be used, and may be selected according to thediscriminatory power desired. Biallelic markers, sets of biallelicmarkers, probes, primers, and methods for determining the identity ofsaid biallelic markers are further described herein.

[0624] Discriminatory Potential of Biallelic Marker Typing

[0625] Calculating Discriminatory Potential

[0626] The discriminatory potential of the forensic test can bedetermined in terms of the profile frequency, also referred to as therandom match probability, by applying the product rule. The product ruleinvolves multiplying the allelic frequencies of all the individualalleles tested, and multiplying by an additional factor of 2 for eachheterozygous locus.

[0627] In one example discussed below, the discriminatory potential ofbiallelic marker typing can be considered in the context of forensicscience. In order to determine the discriminatory potential with respectto the numbers of biallelic markers to be used in a genetic typingsystem, the formulas and calculations below assume that (1) thepopulation under study is sufficiently large (so that we can assume noconsanguinity); (2) all markers chosen are not correlated, so that theproduct rule (Lander and Budlowle (1992)) can be applied; and (3) theceiling rule can be applied or that the allelic frequencies of markersin the population under study are known with sufficient accuracy.

[0628] As noted in Weir, B. S., Genetic data Analysis II: Methods forDiscrete population genetic Data, Sinauer Assoc., Inc., Sunderland,Mass., USA, 1996, the example assumes a crime has been committed and asample of DNA from the perpetrator (P) is available for analysis. Thegenotype of this DNA sample can be determined for several geneticmarkers, and the profile A of the perpetrator can thereby be determined.

[0629] In this example, one suspect (S) is available for typing. Thesame set of genetic markers, such as the biallelic markers of theinvention, are typed and the same profile A is obtained for (S) and (P).Two hypotheses are thus presented as follows:

[0630] (1) either S is P (event C)

[0631] (2) either S is not P (event C).

[0632] The ratio L of both probabilities can then be calculated usingthe following equation:$L = \frac{{pr}\left( {{S = A},{P = {A/C}}} \right)}{{pr}\left( {{S = A},{P = {A/\overset{\_}{C}}}} \right)}$

[0633] L can then further be calculated by the following equation:$\begin{matrix}{L = {\frac{1}{{pr}\left( {{P = {{A/S} = A}},\overset{\_}{C}} \right)}(1)}} & \text{Equation~~1}\end{matrix}$

[0634] These probabilities as well as L can be calculated in severalsettings, notably for different kinship coefficients between P and S fora genetic marker (see Weir, (1996)).

[0635] Assuming that all genetic markers chosen are independent of eachother, the global ratio L for a set of genetic markers will be theproduct over each genetic marker of all L.

[0636] It is further possible to estimate the mean number of biallelicmarkers or VNTRs required to have a ratio L equal to 10⁸ or 10⁶ bycalculating the expectancy of the random variable L using the followingequation:${E(L)} = {\prod\limits_{i = 1}^{N}{{E\left( L_{i} \right)}\quad {where}\quad N\quad {is}\quad {the}\quad {number}\quad {of}\quad {loci}}}$${{E\left( L_{i} \right)} = {\sum\limits_{j = 1}^{G_{i}}{{{pr}\left( {{P = {{A_{ij}/S} = A_{ij}}},\overset{\_}{C}} \right)}.L_{ij}}}},{{where}\quad A_{ij}\quad {is}\quad {the}\quad {genotype}\quad j\quad {at}\quad {the}\quad {ith}\quad {marker}},$

[0637] L_(ij) the ratio associated with such genotype, G_(i) being thenumber of genotypes at locus i.

[0638] From equation 1, it can easily be derived that the expectancy ofL_(i) is G_(i), the number of possible genotypes of this marker.

[0639] The general expectancy for a set of genetic markers can then beexpressed by the following equation: $\begin{matrix}{{E(L)} = {\prod\limits_{i = 1}^{N}G_{i}}} & \text{Equation~~2}\end{matrix}$

[0640] A. Biallelic Marker-Based DNA Typing Systems

[0641] Using the equations described above, it is possible to selectbiallelic marker-based DNA typing systems having a desireddiscriminatory potential.

[0642] Using biallelic markers, E(L) can thus be expressed as 3^(N).When using VNTR-based DNA typing systems, assuming the VNTRs have 10alleles, E(L) can be expressed as 55^(N). Based on these results, thenumber of biallelic markers or VNTRs needed to obtain, in mean, a ratioof at least 10⁶ or 10⁸ can calculated, and are set forth below in Table4. TABLE 4 Marker sets L = 10⁶ L = 10⁸ Biallelic 13 17  5-allele markers(e.g. VNTR) 5 7 10-allele markers (e.g. VNTR) 4 5

[0643] Thus, in a first embodiment, DNA typing systems and methods ofthe invention may comprise genotyping a set of at least 13 or at least17 biallelic markers to obtain a ratio of at least 10⁶ or 10⁸, assuminga flat distribution of L across the biallelic markers. In preferredembodiments, a greater number of biallelic markers is genotyped toobtain a higher L value. Preferably at least 1, 2, 3, 4, 5, 10, 13, 15,17, 20, 25, 30, 40, 50, 70, 85, 100, 150, 200, 300, 400, 500, 600 or allof the eicosanoid-related biallelic markers are genotyped. Said DNAtyping systems of the invention would result in L values as listed inTable 5 below as an indication of the discriminate potential of thesystems of the invention. TABLE 5 Number of biallelic markers L  507.2 * 10²² 100   5 * 10⁴⁷ 650 3{circumflex over ( )}650

[0644] In situations where the distribution of L is not flat, such as inthe worst case when the perpetrator is homozygous for the major alleleat each genetic locus and L thus takes the lowest value, a larger numberof biallelic markers is required for the same discriminatory potential.Therefore, in preferred embodiments, DNA typing systems and methods ofthe invention using a larger number of biallelic markers allow foruneven distributions of L across the biallelic markers. For example,assuming unrelated individuals, a set of independent markers having anallelic frequency of 0.1/0.9, and the genetic profile of a homozygote ateach genetic loci for the major allele, 66 biallelic markers arerequired to obtain a ratio of 10⁶, and 88 biallelic markers are requiredto obtain a ratio of 10⁸. Thus, in preferred embodiments based on theuse of markers having a major allele of sufficiently high frequency,this is a first estimation of the upper bound of markers required in aDNA typing system.

[0645] In further embodiments, it is also desirable to have the abilityto discriminate between relatives. Although unrelated individuals have alow probability of sharing genetic profiles, the probability is greatlyincreased for relatives. For example, the DNA profile of a suspectmatches the DNA profile of a sample at a crime scene, and theprobability of obtaining the same DNA profile if left by an untypedrelative is required. Table 6 below (Weir (1996)) lists probabilitiesfor several different types of relationships, assuming alleles A_(i) andA_(j), and population frequencies P_(i) and p_(j), and lists likelihoodratios assuming genetic loci having allele frequencies of 0.1. TABLE 6Genotype Relationship Pr(p = A|S = A) L A_(i) A_(j) Full brothers (1 +p_(i) + p_(j) + 2p_(i)p_(j))/4 3.3 Father and son (p_(i) + p_(j))/2 10.0Half brothers (p_(i) + p_(j) + 4p_(i)p_(j))/4 16.7 Uncle and nephew (1 +p_(i) + p_(j) + 2p_(i)p_(j))/4 16.7 First cousins (1 + p_(i) + p_(j) +12p_(i)p_(j))/8 25.0 Unrelated 2p_(i)p_(j) 50.0 A_(j) A_(j) Fullbrothers (1 + p_(i))²/4 3.3 Father and son p_(i) 10.0 Half brothersp_(i) (1 + p_(i))/2 18.2 Uncle and nephew p_(i) (1 + p_(i))/2 18.2 Firstcousins p_(i) (1 + 3p_(i))/4 30.8 Unrelated p_(i) ² 100.0

[0646] In one example, where the suspect is the full brother of theperpetrator, the number of required biallelic markers will be 187assuming the profile is that of a homozygote for the major allele ateach biallelic marker.

[0647] In yet further embodiments, the DNA typing systems and methods ofthe present invention may further take into account effects ofsubpopulations on the discriminatory potential. In embodiments describedabove for example, DNA typing systems consider close familialrelationships, but do not take into account membership in the samepopulation. While population membership is expected to have littleeffect, the invention may further comprise genotyping a larger set ofbiallelic markers to achieve higher discriminatory potential.Alternatively, a larger set of biallelic markers may be optimized fortyping selected populations; alternatively, the ceiling principle may beused to study allele frequencies from individuals in various populationsof interest, taking for any particular genotype the maximum allelefrequency found among the populations.

[0648] The invention thus encompasses methods for genotyping comprisingdetermining the identity of a nucleotide at least 13, 15, 17, 20, 25,30, 40, 50, 66, 70, 85, 88, 100, 187, 200, 300, 500, 700, 1000 or 2000biallelic markers in a biological sample, wherein at least 1, 2, 3, 4,5, 10, 13, 17, 20, 25, 30, 40, 50, 70, 85, 100, 150, 200, 300, 400, 500,600 or all of said biallelic markers are eicosanoid-related biallelicmarkers selected from the group consisting of the markers provided inTable 7(A-B).

[0649] Any markers known in the art may be used with theeicosanoid-related biallelic markers of the present invention in the DNAtyping methods and systems described herein, for example in anyone ofthe following web sites offering collections of SNPs and informationabout those SNPs:

[0650] The Genetic Annotation Initiative (http://cap.nci.nih.gov/GAI/).An NIH run site which contains information on candidate SNPs thought tobe related to cancer and tumorigenesis generally.

[0651] dbSNP Polymorphism Repository (ttp://www.ncbi.nlm.nhi.gov/SNP/).A more comprehensive NIH-run database containing information on SNPswith broad applicability in biomedical research.

[0652] HUGO Mutation Database Initiativehttp://aricl.ucs.unimelb.edu.au:80/˜cotton/mdi.htm). A database meant toprovide systematic access to information about human mutations includingSNPs. This site is maintained by the Human Genome Organization (HUGO).

[0653] Human SNP Database(http://www-genome.wi.mit.edu/SNP/human/index.html). Managed by theWhitehead Institute for Biomedical Research Genome Institute, this sitecontains information about SNPs resulting from the many Whiteheadresearch projects on mapping and sequencing.

[0654] SNPs in the Human-Genome SNP database(http://www.ibc.wustl.edu/SNP). This website provides access to SNPsthat have been organized by chromosomes and cytogenetic location. Thesite is run by Washington University.

[0655] HGBase http://hgbase.cgr.ki.se/). HGBASE is an attempt tosummarize all known sequence variations in the human genome, tofacilitate research into how genotypes affect common diseases, drugresponses, and other complex phenotypes, and is run by the KarolinskaInstitute of Sweden.

[0656] The SNP Consortium Database (http://snp.cshl.org/db/snp/map. Acollection of SNPs and related information resulting from thecollaborative effort of a number of large pharmaceutical and informationprocessing companies.

[0657] GeneSNPs (http://www.genome.utah.edu/genesnps/). Run by theUniversity of Utah, this site contains information about SNPs resultingfrom the U. S. National Institute of Environmental Health's initiativeto understand the relationship between genetic variation and response toenvironmental stimuli and xenobiotics.

[0658] In addition, biallelic markers provided in the following patentsand patent applications may also be used with the eicosanoid-relatedbiallelic markers of the invention in the DNA typing methods and systemsdescribed above: U.S. Ser. No. 60/206,615, filed Mar. 24, 2000; U.S.Ser. No. 60/216,745, filed Jun. 30, 2000; WIPO Serial No.PCT/IB00/00184, filed Feb. 11, 2000; WIPO Serial No. PCT/IB98/01193,filed Jul. 17, 1998; PCT Publication No. WO 99/54500, filed Apr. 21,1999; and WIPO Serial No. PCT/IB00/00403, filed Mar. 24, 2000.

[0659] Biallelic markers, sets of biallelic markers, probes, primers,and methods for determining the identity of a nucleotide at saidbiallelic markers are also encompassed and are further described herein,and may encompass any further limitation described in this disclosure,alone or in any combination.

[0660] Forensic matching by microsequencing is further described inExample 8 below.

EXAMPLES

[0661] Several of the methods of the present invention are described inthe following examples, which are offered by way of illustration and notby way of limitation. Many other modifications and variations of theinvention as herein set forth can be made without departing from thespirit and scope thereof and therefore only such limitations should beimposed as are indicated by the appended claims.

Example 1

[0662] De Novo Identification Of Biallelic Markers

[0663] The biallelic markers set forth in this application were isolatedfrom human genomic sequences. To identify biallelic markers, genomicfragments were amplified, sequenced and compared in a plurality ofindividuals.

[0664] DNA Samples

[0665] Donors were unrelated and healthy. They represented a sufficientdiversity for being representative of a French heterogeneous population.The DNA from 100 individuals was extracted and tested for the de novoidentification of biallelic markers.

[0666] DNA samples were prepared from peripheral venous blood asfollows. Thirty ml of peripheral venous blood were taken from each donorin the presence of EDTA. Cells (pellet) were collected aftercentrifugation for 10 minutes at 2000 rpm. Red cells were lysed in alysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mMNaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many timesas necessary to eliminate the residual red cells present in thesupernatant, after resuspension of the pellet in the lysis solution. Thepellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysissolution composed of: (a) 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl0.4 M; (b) 200 μl SDS 10%; and (c) 500 μl proteinase K (2 mg proteinaseK in TE 10-2/NaCl 0.4 M).

[0667] For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5v/v) was added. After vigorous agitation, the solution was centrifugedfor 20 minutes at 10000 rpm. For the precipitation of DNA, 2 to 3volumes of 100% ethanol were added to the previous supernatant, and thesolution was centrifuged for 30 minutes at 2000 rpm. The DNA solutionwas rinsed three times with 70% ethanol to eliminate salts, andcentrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C.,and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration wasevaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA). Todetermine the presence of proteins in the DNA solution, the OD 260/OD280 ratio was determined. Only DNA preparations having a OD 260/OD 280ratio between 1.8 and 2 were used in the subsequent examples describedbelow. DNA pools were constituted by mixing equivalent quantities of DNAfrom each individual.

[0668] Amplification of Genomic DNA by PCR

[0669] Amplification of specific genomic sequences was carried out onpooled DNA samples obtained as described above.

[0670] Amplification Primers

[0671] The primers used for the amplification of human genomic DNAfragments were defined with the OSP software (Hillier & Green, 1991).Preferably, primers included, upstream of the specific bases targetedfor amplification, a common oligonucleotide tail useful for sequencing.Primers PU contain the following additional PU 5′ sequence :TGTAAAACGACGGCCAGT; primers RP contain the following RP 5′ sequence :CAGGAAACAGCTATGACC. Primers are listed in Table 13. Amplification PCRassays were performed using the following protocol: Final volume   25 μlDNA   2 ng/μl MgCl₂   2 mM dNTP (each)  200 μM primer (each)  2.9 ng/μlAmpli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10x = 0.1 MTrisHCl pH 8.3 0.5M KCl) 1x

[0672] DNA amplification was performed on a Genius II thermocycler.After heating at 94° C. for 10 min, 40 cycles were performed. Cyclingtimes and temperatures were: 30 sec at 94° C., 55° C. for 1 min and 30sec at 72° C. Holding for 7 min at 72° C. allowed final elongation. Thequantities of the amplification products obtained were determined on96-well microtiter plates, using a fluorometer and Picogreen asintercalant agent (Molecular Probes).

[0673] Sequencing of Amplified Genomic DNA and Identification ofBiallelic Polymorphisms

[0674] Sequencing of the amplified DNA was carried out on ABI 377sequencers. The sequences of the amplification products were determinedusing automated dideoxy terminator sequencing reactions with a dyeterminator cycle sequencing protocol. The products of the sequencingreactions were run on sequencing gels and the sequences were determinedusing gel image analysis (ABI Prism DNA Sequencing Analysis software2.1.2 version).

[0675] The sequence data were further evaluated to detect the presenceof biallelic markers within the amplified fragments. The polymorphismsearch was based on the presence of superimposed peaks in theelectrophoresis pattern resulting from different bases occurring at thesame position. However, the presence of two peaks can be an artifact dueto background noise. To exclude such an artifact, the two DNA strandswere sequenced and a comparison between the two strands was carried out.In order to be registered as a polymorphic sequence, the polymorphismhad to be detected on both strands. Further, some biallelic singlenucleotide polymorphisms were confirmed by microsequencing as describedbelow.

[0676] Biallelic markers were identified in the analyzed fragments andare shown in Table 7. Also, the genomic structure of the FLAP gene and12-LO gene including the relative location of some biallelic markers isshown in FIG. 1 and FIG. 3, respectively.

Example 2

[0677] Genotyping of Biallelic Markers

[0678] The biallelic markers identified as described above were furtherconfirmed and their respective frequencies were determined throughmicrosequencing. Microsequencing was carried out on individual DNAsamples obtained as described herein.

[0679] Microsequencing Primers

[0680] Amplification of genomic DNA fragments from individual DNAsamples was performed as described in Example 1 using the same set ofPCR primers. Microsequencing was carried out on the amplified fragmentsusing specific primers. See Table 12. The preferred primers used inmicrosequencing had about 19 nucleotides in length and hybridized justupstream of the considered polymorphic base.

[0681] The microsequencing reactions were performed as follows: 5 μl ofPCR products were added to 5 μl purification mix (2U SAP (Shrimpalkaline phosphate) (Amersham E70092X)); 2U Exonuclease I (AmershamE70073Z); and 1 μl SAP buffer (200 MM Tris-HCI pH8, 100 mM MgCI₂) in amicrotiter plate. The reaction mixture was incubated 30 minutes at 37°C., and denatured 10 minutes at 94° C. afterwards. To each well was thenadded 20 μl of microsequencing reaction mixture containing: 10 pmolmicrosequencing oligonucleotide (19 mers, GENSET, crude synthesis, 5OD), 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenasebuffer (260 mM Tris HCl pH 9.5, 65 MM MgCl₂), and the two appropriatefluorescent ddNTPs complementary to the nucleotides at the polymorphicsite corresponding to both polymorphic bases (11.25 nM TAMRA-ddTTP;16.25 nM ROX-ddCTP; 1.675 nM REG-ddATP; 1.25 nM RHO-ddGTP ; PerkinElmer, Dye Terminator Set 401095). After 4 minutes at 94° C., 20 PCRcycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. werecarried out in a Tetrad PTC-225 thermocycler (MJ Research). Themicrotiter plate was centrifuged 10 sec at 1500 rpm. The unincorporateddye terminators were removed by precipitation with 19 μl MgCl₂ 2 mM and55 μl 100% ethanol. After 15 minute incubation at room temperature, themicrotiter plate was centrifuged at 3300 rpm 15 minutes at 4° C. Afterdiscarding the supernatants, the microplate was evaporated to drynessunder reduced pressure (Speed Vac). Samples were resuspended in 2.5 μlformamide EDTA loading buffer and heated for 2 min at 95° C. 0.8 μlmicrosequencing reaction were loaded on a 10% (19:1) polyacrylamidesequencing gel. The data were collected by an ABI PRISM 377 DNAsequencer and processed using the GENESCAN software (Perkin Elmer).

[0682] Frequency of Biallelic Markers

[0683] Frequencies are reported for the less common allele only and areshown in Table 7.

Example 3

[0684] Association Study Between Asthma and the Biallelic Markers of theFLAP Gene

[0685] Collection of DNA Samples From Case and Control Individuals

[0686] The disease trait followed in this association study was asthma,a disease involving the leukotriene pathway. The asthmatic populationcorresponded to 298 individuals that took part in a clinical study forthe evaluation of the anti-asthmatic drug Zileuton. More than 90% ofthese 298 asthmatic individuals had a Caucasian ethnic background. Thecontrol population was composed of 286 individuals from a random USCaucasian population.

[0687] Genotvoing of Case and Control Individuals

[0688] The general strategy to perform the association studies was toindividually scan the DNA samples from all individuals in each of thepopulations described above in order to establish the allele frequenciesof the above described biallelic markers in each of these populations.

[0689] Allelic frequencies of the above-described biallelic markeralleles in each population were determined by performing microsequencingreactions on amplified fragments obtained by genomic PCR performed onthe DNA samples from each individual. Genomic PCR and microsequencingwere performed as detailed above in Examples 1 and 2 using the describedPCR and microsequencing primers.

[0690] Frequency of the Biallelic Marker Alleles of the FLAP Gene andAssociation with Asthma

[0691] Frequencies of biallelic marker alleles were compared in thecase-control populations described above. The association curve in FIG.2 shows the p-value obtained for each marker and the localization of themarkers in the genomic region harboring the FLAP gene. As shown in FIG.2, the biallelic marker 10-35-390 presented a strong association withasthma, this association being highly significant (pvalue=2.29×10⁻³).The two markers 10-32-357 and 10-33-234 show association when testedindependently. The biallelic marker 10-35/390 is located in the FLAPgene. Therefore, the association studies results show that apolymorphism of the FLAP gene seems to be related to asthma. Thebiallelic marker 10-35-390 can be then used in diagnostics with a testbased on this marker or on a combination of biallelic markers comprisingthis marker.

[0692] Haplotype Frequency Analysis

[0693] The results of the haplotype analysis using 9 biallelic markers(10-253-298, 10-32-357, 10-33-175, 10-33-234, 10-33-327, 10-35-358,10-35-390, 12-628-306, and 12-629-241) are shown in Table 15. Haplotypeanalysis for association of FLAP markers and asthma was performed byestimating the frequencies of all possible 2, 3 and 4 marker haplotypesin the asthmatic and Caucasian US control populations. Haplotypeestimations were performed by applying the Expectation-Maximization (EM)algorithm (Excoffier and Slatkin, 1995), using the EM-HAPLO program(Hawley et al., 1994). Estimated haplotype frequencies in the asthmaticand control populations were compared by means of a chi-squarestatistical test.

[0694] The most significant haplotypes obtained are shown in Table 15.

[0695] Preferred haplotypes comprise either the marker 10-33-234 (alleleA) or the marker 10-35-390 (allele T). Preferred haplotype No. 1 (A at10-33-234 and T at 10-35-390) presented a p-val of 8.2×10⁻⁴ and anodd-ratio of 1.61. Estimated haplotype frequencies were 28.3% in thecases and 19.7% in the US controls. Also preferred are haplotypes No. 2(A at 10-33-234 and G at 12-629-241) and haplotype No. 3 (T at 10-33/327and T at 10-33/390) which presented respectively a p-value of 1.6×10⁻³and 1.8×10⁻³, an odd-ratio of 1.65 and 1.53 and haplotypes frequenciesof 0.305 and 0.307 for the asthmatic population and of 0.210 and 0.224for the US control population.

[0696] Preferred haplotypes consisting of three markers (haplotype nos.37, 38, 39 and 41) comprise the marker 10-33-234 (allele A) and themarker 10-35-390 (allele T). Preferred haplotype No. 37 (A at 10-33-234,T at 10-33-390 and C at 12-628-306) presented a p-value of 8.6×10⁻⁴ andan odd-ratio of 1.76. Estimated haplotype frequencies were 26.5% in thecases and 17.1% in the US controls. Haplotype No. 40 (A at 10-33-234, Cat 12-628-306 and G at 12-629-241) is also very significantly associatedwith asthma.

[0697] Four-marker haplotypes (haplotype Nos. 121 to 125), five-markerhaplotypes (haplotype Nos. 247 and 248) and a six-marker haplotype(haplotype No. 373) also showed significant p-values. They all comprisemarkers 10-33-234 (allele A) and 10-35/390 (allele T), except haplotypeno. 124. Other markers in these haplotypes are chosen from the groupconsisting of 10-235-298 (allele C), 10-35-358 (allele G), 12-628-306(allele C) and 12-629-241 (allele G).

[0698] Haplotype No. 1 is the preferred haplotype of the invention. Itcan be used in diagnosis of asthma. Moreover, most of the haplotypessignificantly associated with asthma comprise the biallelic marker10-35-390 (allele A) and could also be used in diagnosis.

[0699] The statistical significance of the results obtained for thehaplotype analysis was evaluated by a phenotypic permutation testreiterated 1000 or 10,000 times on a computer. For this computersimulation, data from the asthmatic and control individuals were pooledand randomly allocated to two groups which contained the same number ofindividuals as the case-control populations used to produce the datasummarized in Table 15. A haplotype analysis was then run on theseartificial groups for the 2 markers included in the haplotype No. 1,which showed the strongest association with asthma. This experiment wasreiterated 1000 and 10,000 times and the results are shown in Table 16.These results demonstrate that among 1000 iterations none and among10,000 iterations only 1 of the obtained haplotypes had a p-valuecomparable to the one obtained for the haplotype No. 1. These resultsclearly validate the statistical significance of the association betweenthis haplotype and asthma.

Example 4

[0700] Association Between Asthma and the Biallelic Markers of the12-lipooxzyenase Gene

[0701] Collection of DNA Samples From Case and Control Individuals

[0702] The disease trait followed in this association study was asthma,a disease involving the leukotriene pathway. The asthmatic populationcorresponded to 297 individuals that took part in a clinical study forthe evaluation of the anti-asthmatic drug zileuton. More than 90% ofthese 297 asthmatic individuals had a Caucasian ethnic background. Thecontrol population corresponded to 186 individuals from a random USCaucasian population.

[0703] Genotyping of Case and Control Individuals

[0704] The general strategy to perform the association studies was toindividually scan the DNA samples from all individuals in each of thepopulations described above in order to establish the allele frequenciesof the above described biallelic markers in each of these populations.

[0705] Allelic frequencies of the above-described biallelic markeralleles in each population were determined by performing microsequencingreactions on amplified fragments obtained by genomic PCR performed onthe DNA samples from each individual. Genomic PCR and microsequencingwere performed as detailed above in Examples 1 and 2 using the describedPCR and microsequencing primers.

[0706] Haplotype Frequency Analysis

[0707] None of the single marker alleles showed a significantassociation with asthma however, significant results were obtained inhaplotype studies. Allelic frequencies were useful to check that themarkers used in the haplotype studies meet the Hardy-Weinbergproportions (random mating).

[0708] Haplotype analysis was performed using 12 biallelic markers and17 biallelic markers. The results of the haplotype analysis using 12biallelic markers (12-208-35, 12-226-167, 12-206-366, 10-347-203,10-347-220, 10-349-97, 10-349-224, 12-196-119, 12-214-129, 12-216-421,12-219-230 and 12-223-207) are shown in Table 17. The results of thehaplotype analysis using 17 biallelic markers (12-197-244, 12-208-35,12-226-167, 12-206-366, 10-346-141, 10-347-165, 10-347-203, 10-347-220,10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421and 12-219-230) are shown in Table 18. Haplotype analysis forassociation of 12-LO biallelic markers and asthma was performed byestimating the frequencies of all possible 2, 3 and 4 marker haplotypesin the asthmatic and control populations described above. Haplotypeestimations were performed by applying the Expectation-Maximization (EM)algorithm (Excoffier and Slatkin, Mol. Biol. Evol., 12:921-927, 1995),using the EM-HAPLO program (Hawley et al., Am. J. Phys.Anthropol.,18:104, 1994) as described above. Estimated haplotypefrequencies in the asthmatic and control population were compared bymeans of a chi-square statistical test (one degree of freedom).

[0709] Table 17 shows the most significant haplotypes obtained from the12 biallelic marker analysis. Haplotype No. 1 consisting of threebiallelic markers (10-347-220, 12-214-129 and 12-219-230) presented ap-value of 2.10⁻⁵ and an odd-ratio of 3.38. Estimated haplotypefrequencies were 12.3% in the cases and 4% in the controls. HaplotypeNo.14 consisting of four biallelic markers (10-347-203, 12-196-119,12-216-421 and 12-219-230) had a p-value of 4.10⁻⁶ and an odd ratio of4.18. Estimated haplotype frequencies were 11.8% in the cases and 3.1%in the controls. Haplotype No.1 and haplotype No.14, are both stronglyassociated with asthma. Haplotypes Nos. 2-13 and 15-24 also showed verysignificant Association (see Table 17).

[0710] Table 18 shows the most significant haplotypes obtained from the17 biallelic marker analysis. Haplotype No. 1 consisting of twobiallelic markers (12-206-366 and 10-349-224) presented a p-value of 1.810⁻⁴ and an odd-ratio of 2.05. Estimated haplotype frequencies were42.4% in the cases and 26.5% in the controls. Haplotype No. 7 consistingof three biallelic markers (10-349-97, 12-214-129, 12-219-230) had ap-value of 2.3 10-5 and an odd ratio of 3.32. Estimated haplotypefrequencies were 12.5% in the cases and 4.1% in the controls. HaplotypeNo. 27 consisting of four biallelic markers (10-349-97, 12-196-119,12-216-421 and 12-219-230) had a p-value of 5.4 10⁻⁶ and an odd ratio of3.90. Estimated haplotype frequencies were 12.4% in the cases and 3.5%in the controls. Haplotypes Nos. 1, 7 and 27 are strongly associatedwith asthma. Other haplotypes also showed very significant association(see Table 18).

[0711] The statistical significance of the results obtained for thehaplotype analysis was evaluated by a phenotypic permutation testreiterated 1000 or 10,000 times on a computer. For this computersimulation, data from the asthmatic and control individuals were pooledand randomly allocated to two groups which contained the same number ofindividuals as the case-control populations used to produce the datasummarized in Tables 17 and 18. A haplotype analysis was then run onthese artificial groups for the markers included in haplotype No. 14from Table 17 and for the markers included in haplotypes Nos. 7 and 27from Table 18, which showed the strongest association with asthma. Thisexperiment was reiterated 1000 and 10,000 times and the results areshown in Table 21 and Table 22, respectively. These results demonstratethat among 1000 iterations only 7 and among 10,000 iterations only 39 ofthe obtained haplotypes from the 12 biallelic marker set had a p-valuecomparable to the one obtained for haplotype No.14 from Table 17. Also,among 1000 iterations only 2 of the obtained haplotypes from the 17biallelic marker set had a p-value comparable to the one obtained forhaplotype No. 7 from Table 18. These results further demonstrate thatamong 1000 iterations none of the obtained haplotypes had a p-valuecomparable to the one obtained for haplotype No. 27 from Table 18. Theseresults clearly validate the statistical significance of the associationbetween the haplotypes shown in Tables 17 and 18 and asthma.

Example 5

[0712] Association Between Side Effects upon Treatment with theAnti-Asthmatic Drug Zileuton (Zyflo™) and the Biallelic Markers of the12-lipoxygenase Gene

[0713] Collection of DNA Samples From Case and Control Individuals

[0714] The side effect examined in this study was the hepatotoxicityexperienced by asthmatic individuals as a result of their treatment withZileuton as part of a clinical study. Asthmatic individuals wereunrelated and more than 90% of the individuals had a Caucasian ethnicbackground. Hepatotoxicity was monitored by measuring the serum levelsof alanine aminotransferase (ALT), which is a sensitive indicator ofliver cell damage.

[0715] More than 90% of the asthmatic individuals participating in thisstudy did not experience Zileuton-associated ALT increase compared totheir ALT levels prior to zileuton intake. As mentioned above, anassociation study is more informative if the case-control populationspresent extreme phenotypes. Therefore, the asthmatic individuals, whichwere selected for the side effect positive trait (ALT+), corresponded to89 individuals that presented at least 3 times the upper limit of normal(ULN) level of ALT. On the other side, the asthmatic individuals thatwere selected for the side effect negative trait (ALT−) corresponded to208 individuals that presented less than 1×ULN of ALT. ALT+ andALT−populations corresponded to 4% and 35% respectively of the totalasthmatic individuals that participated in this study.

[0716] Genotyping of Case and Control Individuals

[0717] The general strategy to perform the association studies was toindividually scan the DNA samples from all individuals in each of thepopulations described above in order to establish the allele frequenciesof the above described biallelic markers in each of these populations.

[0718] Allelic frequencies of the above-described biallelic markeralleles in each population were determined by performing microsequencingreactions on amplified fragments obtained by genomic PCR performed onthe DNA samples from each individual. Genomic PCR and microsequencingwere performed as detailed above in Examples 1 and 2 using the describedPCR and microsequencing primers.

[0719] Haplotype Frequency Analysis

[0720] None of the single marker alleles showed a significantassociation with hepatoxicity to zileuton, however, significant resultswere obtained in haplotype studies.

[0721] Haplotype analysis was performed using 12 biallelic markers and17 biallelic markers. The results of the haplotype analysis using 12biallelic markers (12-208-35, 12-226-167, 12-206-366, 10-347-203,10-347-220, 10-349-97, 10-349-224, 12-196-119, 12-214-129, 12-216-421,12-219-230 and 12-223-207) are shown in Table 19. The results of thehaplotype analysis using 17 biallelic markers (12-197-244, 12-208-35,12-226-167, 12-206-366, 10-346-141, 10-347-165, 10-347-203, 10-347-220,10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421and 12-219-230) are shown in Table 20. Haplotype analysis forassociation of 12-LO biallelic markers and asthma was performed byestimating the frequencies of all possible 2, 3, 4 and 5 markerhaplotypes in the ALT+ and ALT−populations described above. Haplotypeestimations were performed by applying the Expectation-Maximization (EM)algorithm (Excoffier and Slatkin, Mol. Biol. Evol., 12:921-927, 1995),using the EM-HAPLO program (Hawley et al., Am. J. Phys.Anthropol.,18:104, 1994) as described above. Estimated haplotypefrequencies in the ALT+ and ALT−populations were compared by means of achi-square statistical test (one degree of freedom).

[0722] Table 19 shows the most significant haplotypes obtained from the12 biallelic marker analysis. Haplotype No.3 consisting of threebiallelic markers (10-349-224, 12-216-421 and 12-223-207) presented ap-value of 4.10⁻⁵ and an odd-ratio of 3.53. Estimated haplotypefrequencies were 15.1% in the cases and 4.8% in the controls. HaplotypeNo. 8 consisting of four biallelic markers (12-206-366, 10-349-224,12-216-421 and 12-223-207) had a p-value of 2.9.10⁻⁶ and an odd ratio of4.56. Estimated haplotype frequencies were 15.8% in the cases and 4% inthe controls. Both haplotypes showed strong association with elevatedserum ALT level upon treatment with zileuton. Both haplotypes arerelated as three out of four biallelic marker alleles (T at 10-349-224,A at 12-216-421 and T at 12-223-207) are common to both haplotypes.Haplotypes Nos. 4-7 and 9-25 also showed very significant association.

[0723] Table 20 shows the most significant haplotypes obtained from the17 biallelic marker analysis. Haplotype No. 11 consisting of threebiallelic markers (12-197/244, 10-349-224 and 12-216-421) presented ap-value of 1.7.10⁻³ and an odd-ratio of 2.66, for alleles CTArespectively. Estimated haplotype frequencies were 13.7% in the casesand 5.6% in the controls. The p-value obtained by a chi-squaredistribution with 7 df for this combination of markers is 2.310⁻² byOmnibus test suggesting that result is highly significant. Anotherhaplotype consisting of four biallelic markers (12-208-35,10-512/36,12-196-119 and 12-219/230) presented a p-value of 3.7.10⁻⁵ andan odd-ratio of 3.74. Estimated haplotype frequencies were 14.7% in thecases and 4.4% in the controls. The p-value obtained by a chi-squaredistribution with 15 df for this combination of markers is 5.410⁻⁴ byOmnibus test. Both haplotypes showed strong association with elevatedserum ALT level upon treatment with zileuton. Both haplotypes arerelated as three out of four biallelic marker alleles (C at 12-197/244,T at 10-349-224 and A at 12-216-421) are common to both haplotypes.Other haplotypes also showed very significant association.

[0724] The statistical significance of the results obtained for thehaplotype analysis was evaluated by a phenotypic permutation testreiterated 100, 1000 or 10,000 times on a computer. For this computersimulation, data from the ALT+ and ALT−populations were pooled andrandomly allocated to two groups which contained the same number ofindividuals as the ALT+ and ALT−populations used to produce the datasummarized in Tables 19 and 20. A haplotype analysis was then run on theartificial groups for the 4 markers included in haplotype No. 8 fromTable 17 and on the artificial groups for the 4 markers included inhaplotype No. 13 from Table 18, which showed the strongest associationwith secondary effects to zileuton. This experiment was reiterated 1000and 10,000 times and the results are shown in Table 21 and Table 22,respectively. These results demonstrate that among 1000 iterations only5 and among 10,000 iterations only 77 of the obtained haplotypes fromthe 12 biallelic markers had a p-value comparable to the one obtainedfor haplotype No. 8. These results demonstrate that among 100 iterationsonly 3 of the obtained haplotypes from the 17 biallelic markers had ap-value comparable to the one obtained for haplotype No. 11. The p-valueobtained by permutating affected status for the omnibus LR test is2.2.10⁻². These results clearly validate the statistical significance ofthe association between hepatotoxicity to Zyflo™ and the haplotypes Nos.3-25 and Nos. 6-30 shown in Table 19 and Table 20, respectively.

[0725] Allele Frequency Analysis

[0726] Allele frequencies were determined in a random US caucasianpopulation, in an asthmatic population showing no side effects upontreatment with Zyflo™ (ALT−) and in an asthmatic population showingelevated alanine aminotransferase levels upon treatment with Zyflo™(ALT+). Table 23 is a chart containing a list of preferred 12-LO-relatedbiallelic markers with an indication of the frequency of the leastcommon allele determined by genotyping as described in Example 2.

Example 6

[0727] Identification Of Mutations And Of Low Frequency Alleles Of The12-LO Gene

[0728] Exons 6, 8 and 14 of the 12-lipoxygenase gene were screened formutations by comparing their sequence in individuals exhibiting elevatedALT levels upon treatment with zileuton (ALT+) and in individualsshowing normal ALT levels upon treatment with zileuton (ALT−). ALT+ andALT−individuals are further described in Example 5. Intron sequencesimmediately flanking these exons were also screened.

[0729] To identify mutations, fragments of the 12-LO gene wereamplified, sequenced and compared in ALT+ and ALT−individuals. DNAsamples from each individual were processed separately.

[0730] DNA Samples

[0731] Individual DNA samples were obtained as described in Example 1.

[0732] Amplification of the 12-LO Gene

[0733] Amplification primers are described in Table 13. PCR assays wereperformed as described in Example 1.

[0734] Sequencing of Amplified Genomic DNA: Identification of Mutationsand of Low Frequency Polymorphisms

[0735] Sequencing of the amplified DNA was carried out on ABI 377sequencers. The sequences of the amplification products were determinedusing automated dideoxy terminator sequencing reactions with a dyeterminator cycle sequencing protocol. The products of the sequencingreactions were run on sequencing gels and the sequences were determinedusing gel image analysis (ABI Prism DNA Sequencing Analysis software2.1.2 version).

[0736] The sequence data was further analyzed to detect the presence ofmutations and of low frequency alleles. The sequences of exon 6, exon 8,exon 14 and flanking intronic sequences in 79 ALT+individuals and 105ALT−individuals were compared. New polymorphisms/mutations were detectedand the genotype of each individual for these markers was determined.Results are shown below: Position in 12-LO Least Common Original MarkerID Gene Allele/Mutation Allele 10-508-191 5′ flanking region C T10-508-245 5′ flanking region T C 10-511-62 5′ flanking region T C10-511-337 5′ flanking region Insertion T — 10-512-36 5′ flanking regionC G 10-512-318 5′ flanking region A G 10-513-250 5′ flanking region A G10-513-262 5′ flanking region T C 10-513-352 5′ flanking region A G10-513-365 5′ flanking region A G 10-343-231 Exon 2 Deletion C —10-343-366 Intron 2 C T 10-343-278 Intron 2 T C 10-343-339 Intron 4 T G10-346-23 Intron 4 G A 10-346-141 Exon 5 A G 10-346-263 Intron 5 G C10-346-305 Intron 5 C T 10-347-74 Intron 5 A G 10-347-111 Exon 6 G C10-347-165 Exon 6 T C 10-347-203 Exon 6 G A 10-347-220 Exon 6 A G10-347-271 Intron 6 T A 10-347-348 Intron 6 A G 10-348-391 Intron 7 A G10-349-47 Intron 7 C T 10-349-97 Exon 8 G A 10-349-142 Exon 8 G C10-349-216 Exon 8 Deletion CTG — 10-349-224 Exon 8 T G 10-349-368 Intron8 C T 10-350-72 Intron 8 T C 10-350-332 Intron 9 C T 10-507-170 Exon 11G A 10-507-321 Intron 11 A C 10-507-353 Intron 11 T C 10-507-364 Intron11 T C 10-507-405 Intron 11 T C 10-339-32 Intron 11 T C 10-339-124Intron 11 T C 10-340-112 Exon 13 A C 10-340-130 Exon 13 A T 10-340-238Intron 13 A G 10-341-116 Exon 14 A G 10-341-319 Exon 14 T C (5′UTR)10-342-301 3′ flanking region Insertion A — 10-342-373 3′ flankingregion T C

[0737] Low frequency polymorphisms and mutations identified in exons 5,6, 8, and 13 are associated with amino acid substitutions at thepolypeptide level. In each of these amino acid substitutions theoriginal residue is replaced by a non-equivalent amino acid presentingdifferent chemical properties. As a consequence, specificity, activityand function of the 12-LO enzyme are modified. Biallelic marker10-343-231 is associated with a frame shift in the open reading frame ofthe 12-LO gene leading to the expression of a variant 12-LO polypeptidecomprising only 131 amino acids. This mutant 12-LO enzyme is probablyinactive or shows differences in specificity, activity and function.Biallelic marker 10-343-231 is associated with the deletion of a Leuresidue in the 12-LO polypeptide.

[0738] The mutations and low frequency polymorphisms listed aboverepresent potential functional mutations of the 12-LO gene.

Example 7

[0739] Preparation of Antibody Compositions to 12-Lipoxygenase Variants

[0740] Preferably antibody compositions, specifically binding the189-His variant of the 12-LO protein or, to the 225-His variant of the12-LO protein or, to the 243-Cys variant of the 12-LO protein or, to the261-Arg variant of the 12-LO protein or, to the 322-Asn variant of the12-LO or, to the 337-Arg variant of the 12-LO protein or to the 574-Lysvariant of 12-LO, are prepared. Other preferred antibody compositions ofthe invention are capable of specifically binding to amino acidpositions 110-131 of SEQ ID No. 654.

[0741] Substantially pure protein or polypeptide is isolated fromtransfected or transformed cells containing an expression vectorencoding the 12-LO protein or a portion thereof. The concentration ofprotein in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms per ml. Monoclonal or polyclonal antibodies to the proteincan then be prepared as follows:

[0742] Monoclonal Antibody Production by Hybridoma Fusion

[0743] Monoclonal antibody to epitopes in the 12-LO protein or a portionthereof can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature, 256:495, 1975, thedisclosure of which is incorporated herein by reference in its entirety)or derivative methods thereof (see Harlow and Lane, Antibodies ALaboratory Manual, Cold Spring Harbor Laboratory, pp. 53-242, 1988, thedisclosure of which is incorporated herein by reference in itsentirety).

[0744] Briefly, a mouse is repetitively inoculated with a few microgramsof the 12-LO protein or a portion thereof over a period of a few weeks.The mouse is then sacrificed, and the antibody producing cells of thespleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, E., Meth. Enzymol. 70:419 (1980), , the disclosure of which isincorporated herein by reference in its entirety, and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Davis, L. et al. Basic Methods inMolecular Biology Elsevier, New York. Section 21-2, the disclosure ofwhich is incorporated herein by reference in its entirety.

[0745] Polyclonal Antibody Production by Immunization

[0746] Polyclonal antiserum containing antibodies to heterogeneousepitopes in the 12-LO protein or a portion thereof can be prepared byimmunizing suitable non-human animal with the 12-LO protein or a portionthereof, which can be unmodified or modified to enhance immunogenicity.A suitable non-human animal is preferably a non-human mammal isselected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, acrude preparation which, has been enriched for 12-LO concentration canbe used to generate antibodies. Such proteins, fragments or preparationsare introduced into the non-human mammal in the presence of anappropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which isknown in the art. In addition the protein, fragment or preparation canbe pretreated with an agent which will increase antigenicity, suchagents are known in the art and include, for example, methylated bovineserum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surfaceantigen, and keyhole limpet hemocyanin (KLH). Serum from the immunizedanimal is collected, treated and tested according to known procedures.If the serum contains polyclonal antibodies to undesired epitopes, thepolyclonal antibodies can be purified by immunoaffinity chromatography.

[0747] Effective polyclonal antibody production is affected by manyfactors related both to the antigen and the host species. Also, hostanimals vary in response to site of inoculations and dose, with bothinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appears to be most reliable. Techniques for producingand processing polyclonal antisera are known in the art, see forexample, Mayer and Walker (1987), the disclosure of which isincorporated herein by reference in its entirety. An effectiveimmunization protocol for rabbits can be found in Vaitukaitis, J. et al.J. Clin. Endocrinol. Metab. 33:988-991 (1971), the disclosure of whichis incorporated herein by reference in its entirety. Booster injectionscan be given at regular intervals, and antiserum harvested when antibodytiter thereof, as determined semi-quantitatively, for example, by doubleimmunodiffusion in agar against known concentrations of the antigen,begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in:Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973), thedisclosure of which is incorporated herein by reference in its entirety.Plateau concentration of antibody is usually in the range of 0.1 to 0.2mg/ml of serum (about 12 μM). Affinity of the antisera for the antigenis determined by preparing competitive binding curves, as described, forexample, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2dEd. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington,D.C. (1980), the disclosure of which is incorporated herein by referencein its entirety.

[0748] Antibody preparations prepared according to either the monoclonalor the polyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

Example 8

[0749] Forensic Matching by Microsequencing

[0750] DNA samples are isolated from forensic specimens of, for example,hair, semen, blood or skin cells by conventional methods. A panel of PCRprimers based on a number of the sequences of of the invention is thenutilized according to the methods described herein to amplify DNA ofapproximately 500 bases in length from the forensic specimen. Thealleles present at each of the selected biallelic markers site accordingto biallelic markers of the invention are then identified accordingExamples discussed herein. A simple database comparison of the analysisresults determines the differences, if any, between the sequences from asubject individual or from a database and those from the forensicsample. In a preferred method, statistically significant differencesbetween the suspect's DNA sequences and those from the sampleconclusively prove a lack of identity. This lack of identity can beproven, for example, with only one sequence. Identity, on the otherhand, should be demonstrated with a large number of sequences, allmatching. Preferably, a minimum of 13, 17, 20, 25, 30, 40, 50, 66, 70,85, 88, 100, 187, 200 or 500 biallelic markers are used to test identitybetween the suspect and the sample.

[0751] The disclosures of all issued patents, published PCTapplications, scientific references or other publications cited hereinare incorporated herein by reference in their entireties.

[0752] Although this invention has been described in terms of certainpreferred embodiments, other embodiments which will be apparent to thoseof ordinary skill in the art of view of the disclosure herein are alsowithin the scope of this invention. Accordingly, the scope of theinvention is intended to be defined only by reference to the appendedclaims.

[0753] In accordance with the regulations relating to Sequence Listings,the following codes have been used in the Sequence Listing to indicatethe locations of biallelic markers within the sequences and to identifyeach of the alleles present at the polymorphic base. The code “r” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is an adenine. The code “y” in thesequences indicates that one allele of the polymorphic base is athymine, while the other allele is a cytosine. The code “m” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is a cytosine. The code “k” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a thymine. The code “s” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a cytosine. The code “w” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is a thymine.

[0754] In some instances, the polymorphic bases of the biallelic markersalter the identity of amino acids in the encoded polypeptide. This isindicated in the accompanying Sequence Listing by use of the featureVARIANT, placement of a Xaa at the position of the polymorphic aminoacid, and definition of Xaa as the two alternative amino acids. Forexample, if one allele of a biallelic marker is the codon CAC, whichencodes histidine, while the other allele of the biallelic marker isCAA, which encodes glutamine, the Sequence Listing for the encodedpolypeptide will contain an Xaa at the location of the polymorphic aminoacid. In this instance, Xaa would be defined as being histidine orglutamine.

[0755] In other instances, Xaa may indicate an amino acid whose identityis unknown because of nucleotide sequence ambiguity. In this instance,the feature UNSURE is used, Xaa is placed at the position of the unknownamino acid, and Xaa is defined as being any of the 20 amino acids or alimited number of amino acids suggested by the genetic code. TABLE 7AList of all of the eicosanoid-related biallelic markers. BIALLELICMARKER VALIDATION GENOTYPING BIALLELIC SEQ ID POSITION IN MICRO- LEASTCOMMON ALLELE GENE MARKER ID NO. SEQ ID NO. SEQUENCING FREQUENCY % FLAP10-253-118 1 478 N FLAP 10-253-298 2 478 Y G 4.57 FLAP 10-253-315 3 478N FLAP 10-499-155 4 478 N FLAP 10-520-256 5 478 N T 40.8 FLAP 10-500-2586 478 N FLAP 10-500-410 7 478 N FLAP 10-503-159 8 478 N FLAP 10-504-1729 478 N FLAP 10-504-243 10 478 N FLAP 10-204-326 11 478 Y A 6.63 FLAP10-32-357 12 478 Y A 33.5 FLAP 10-33-175 13 478 Y T 2.30 FLAP 10-33-21114 478 N FLAP 10-33-234 15 478 Y A 44.0 FLAP 10-33-270 16 478 Y G/G FLAP10-33-327 17 478 Y C 24.5 FLAP 10-34-290 18 478 N FLAP 10-35-358 19 478Y C 31.3 FLAP 10-35-390 20 478 Y T 23.0 FLAP 10-36-164 21 478 Y G/G FLAP10-498-192 22 478 N FLAP 12-628-306 23 478 Y T 10.3 FLAP 12-628-311 24478 N FLAP 12-629-241 25 478 Y C 28.3 12-LO 12-206-366 26 478 Y C 38.212-LO 10-343-339 27 478 N 12-LO 10-347-74 28 478 N 12-LO 10-347-111 29478 N G/G 12-LO 10-347-165 30 478 N C/C 12-LO 10-347-203 31 478 Y G 41.612-LO 10-347-220 32 478 Y A 40.5 12-LO 10-347-271 33 478 N 12-LO10-347-348 34 478 N 12-LO 10-348-391 35 478 N 12-LO 10-349-47 36 478 N12-LO 10-349-97 37 478 Y G 39.6 12-LO 10-349-142 38 478 N C/C 12-LO10-349-224 39 478 Y T 39.6 12-LO 10-349-368 40 478 N 12-LO 10-339-32 41478 N 12-LO 10-341-116 42 478 Y A 10.8 12-LO 10-341-319 43 478 N 12-LO12-196-119 44 119 Y C 29.1 12-LO 12-197-244 45 243 Y C 32.8 12-LO12-198-128 46 128 N 12-LO 12-206-81 47 478 N 12-LO 12-208-35 48 35 Y A42.3 12-LO 12-214-129 49 129 Y C 38.7 12-LO 12-214-151 50 151 N 12-LO12-214-360 51 358 N 12-LO 12-215-467 52 466 N 12-LO 12-216-421 53 418 YA 36.0 12-LO 12-219-230 54 229 Y G 32.1 12-LO 12-219-256 55 255 N 12-LO12-220-48 56 478 N 12-LO 12-221-302 57 302 N 12-LO 12-223-179 58 179 N12-LO 12-223-207 59 207 Y C 38.4 12-LO 12-225-541 60 540 Y C 37.4 12-LO12-226-167 61 166 Y G 41.2 12-LO 12-226-458 62 455 N 12-LO 12-229-332 63332 N 12-LO 12-229-351 64 351 N 12-LO 12-230-364 65 364 N 12-LO12-231-100 66 99 N 12-LO 12-231-148 67 147 N 12-LO 12-231-266 68 265 NcPLA₂ 10-231-23 69 500 Y A 8.79 cPLA₂ 10-233-386 70 501 Y G 28.3 cPLA₂10-239-368 72 501 N cPLA₂ 10-223-30 73 501 Y G 22.5 cPLA₂ 10-223-72 74501 N cPLA₂ 10-223-130 75 501 N cPLA₂ 10-223-262 76 501 N cPLA₂10-223-392 77 501 N cPLA₂ 10-224-341 78 501 N cPLA₂ 10-227-282 79 501 YG 3.93 ANX1 10-240-241 80 501 N ANX1 10-249-185 81 501 N ANX1 10-251-12882 501 N ANX1 10-252-209 83 501 N ANX1 12-387-32 84 501 Y G 33.9 ANX110-242-316 85 500 N ANX1 10-245-412 86 501 N ANX1 12-378-171 87 501 NANX1 12-378-228 88 501 N ANX1 12-378-450 89 501 N ANX1 12-379-65 90 501N ANX1 12-382-204 91 501 Y G 50.0 ANX1 12-383-117 92 501 N ANX112-383-170 93 501 N ANX1 12-383-268 94 501 N ANX1 12-384-336 95 501 NANX1 12-384-451 96 501 N ANX1 12-385-123 97 258 N ANX1 12-385-427 98 501N ANX1 12-386-155 99 443 Y G 8.15 ANX1 12-386-24 100 313 N ANX112-387-177 101 501 Y T 33.5 ANX1 12-389-431 102 501 N ANX1 12-391-366103 501 N ANX1 12-394-85 104 501 N ANX1 12-395-382 105 385 N ANX112-400-217 106 501 Y G 27.2 ANX1 12-400-280 107 501 N ANX1 12-401-378108 380 N ANX1 12-402-126 109 323 N ANX1 12-404-265 110 317 N ANX112-406-52 111 501 N ANX1 12-406-409 112 501 N ANX1 12-407-217 113 501 NANX1 12-407-399 114 501 N ANX1 12-408-355 115 501 Y G 2.69 ANX112-409-221 116 229 N ANX1 12-410-301 117 486 N ANX2 10-395-101 118 501 NANX2 10-395-124 119 501 N ANX2 10-395-155 120 501 N ANX2 10-395-294 121501 N ANX2 10-396-100 122 501 N ANX2 10-397-201 123 501 N ANX210-399-178 124 501 N ANX2 10-400-369 125 501 N ANX2 10-392-20 126 497 NANX2 10-392-103 127 501 N ANX2 10-392-324 128 501 N ANX2 10-393-27 129501 N ANX2 10-393-324 130 501 N ANX2 12-727-237 131 501 N ANX212-728-224 132 501 N ANX2 12-730-142 133 501 N ANX2 12-730-193 134 501 NANX2 12-731-60 135 501 N ANX2 12-731-119 136 501 N ANX2 12-731-137 137501 N ANX2 12-731-146 138 501 N ANX2 12-731-398 139 501 N ANX212-732-113 140 501 N ANX2 12-732-164 141 501 N ANX2 12-732-165 142 501 YC 27.4 ANX2 12-732-445 143 501 N ANX2 12-734-201 144 501 N ANX212-735-42 145 501 N ANX2 12-736-363 146 501 N ANX2 12-737-69 147 501 Y A36.8 ANX2 12-737-296 148 501 N ANX2 12-738-429 149 501 Y T 35.5 ANX212-740-112 150 501 Y G 37.6 ANX2 12-740-118 151 501 N ANX2 12-741-265152 501 N ANX2 12-741-327 153 501 N ANX2 12-741-376 154 501 N ANX212-745-30 155 501 N ANX2 12-745-75 156 501 N ANX2 12-745-343 157 501 NANX2 12-745-350 158 501 N ANX2 12-746-320 159 501 N ANX2 12-747-181 160501 N ANX2 12-747-302 161 501 N ANX2 12-749-240 162 501 N ANX212-749-255 163 501 N ANX2 12-752-37 164 508 N ANX2 12-752-85 165 501 NANX2 12-752-196 166 501 N ANX2 12-752-484 167 501 N ANX2 12-753-139 168501 N ANX2 12-753-376 169 501 N ANX2 12-754-172 170 501 N ANX212-754-218 171 501 N ANX2 12-754-328 172 501 N ANX2 12-754-396 173 501 NANX2 12-755-280 174 501 N ANX2 12-757-384 175 501 N ANX2 12-758-257 176501 N ANX2 12-758-374 177 501 N ANX2 12-758-424 178 501 N ANX2 12-761-23179 541 N ANX2 12-761-178 180 501 N ANX2 12-764-329 181 501 N ANX212-764-377 182 501 N ANX2 12-765-168 183 501 N ANX2 12-765-504 184 501 NANX3 10-372-279 185 501 N ANX3 10-375-136 186 501 N ANX3 10-376-281 187501 N ANX3 10-369-392 188 501 N ANX3 10-371-257 189 501 N ANX312-513-389 190 501 N ANX3 12-513-494 191 501 N ANX3 12-515-394 192 501 NANX3 12-516-97 193 501 Y T 37.2 ANX3 12-520-287 194 501 N ANX312-520-323 195 501 Y A 21.5 ANX3 12-523-179 196 501 Y A 29.9 ANX312-523-270 197 501 N ANX3 12-527-367 198 501 Y T 18.9 ANX3 12-529-376199 501 N ANX3 12-529-489 200 501 N ANX3 12-530-134 201 501 Y T 39.3ANX3 12-530-393 202 501 N ANX3 12-531-173 203 501 Y C 37.6 ANX312-539-441 204 501 N ANX3 12-543-78 205 501 N ANX3 12-543-79 206 501 NANX3 12-546-235 207 501 N ANX3 12-549-287 208 501 N ANX3 12-550-287 209501 N ANX3 12-552-175 210 501 N ANX3 12-554-330 211 501 N ANX312-556-312 212 501 N ANX3 12-556-443 213 501 N ANX3 12-558-205 214 501 NANX3 12-558-238 215 501 N ANX3 12-558-305 216 501 N ANX3 12-769-39 217501 N ANX3 12-769-430 218 501 N ANX3 12-770-73 219 501 N ANX3 12-772-200220 501 N ANX3 12-772-254 221 501 N CAL1 10-87-73 222 72 N CAL1 10-87-74223 73 N CAL1 10-87-80 224 79 N CAL1 10-87-140 225 138 N CAL1 10-88-81226 81 Y C 44.7 CAL1 10-89-41 227 41 N CAL1 10-90-35 228 35 Y A 1.14CAL1 10-91-274 229 274 N CAL1 10-93-133 230 133 N CAL1 10-94-197 231 197Y G/G CAL1 10-94-198 232 198 N CAL1 10-166-362 233 362 N CAL2 10-207-386234 387 Y C/C CAL2 10-207-409 235 409 Y G 9.04 CAL2 10-118-307 236 307 YA 0.27 CAL2 10-173-247 237 247 N CAL2 10-173-294 238 294 Y G 2.87 CAL210-173-347 239 347 Y C/C CAL3 10-103-104 240 104 N CAL3 10-103-323 241323 Y T 22.3 CAL3 10-103-402 242 403 N CAL3 10-106-98 243 98 N CAL310-106-288 244 288 Y CAL3 10-106-378 245 380 Y CAL3 10-168-160 246 160 YT 42.1 CAL3 10-168-206 247 206 Y CAL3 10-168-284 248 283 N CAL310-169-318 249 317 N CALPA1 12-86-79 250 501 Y C 37.4 CALPA1 12-88-393251 501 N CALPA1 12-89-369 252 501 Y G 36.3 CALPA1 12-89-91 253 501 NCALPA1 12-94-210 254 501 N CALPA1 12-94-516 255 521 N CALPA1 12-96-64256 501 Y T 8.52 CALPA1 12-97-83 257 501 N CALPA1 12-99-296 258 501 Y T6.45 CALPA1 12-100-266 259 501 Y G 32.2 CALPA1 12-811-174 260 501 NCALPA1 12-815-94 261 501 N CALPA1 12-815-383 262 501 N CALPA1 12-815-384263 500 N CALPA1 12-815-391 264 501 N CALPA1 12-817-214 265 501 N CALPA112-817-355 266 501 N CALPA1 12-819-437 267 501 N CALPA1 12-821-62 268501 N CALPA1 12-821-483 269 501 N CALPA1 12-825-173 270 501 N CALPA112-826-312 271 501 N CALPA1 12-831-59 272 501 N CALPA1 12-833-264 273501 N CALPA1 12-833-279 274 501 N CALPA1 12-833-280 275 502 N CALPA112-833-373 276 501 N CALPA1 12-834-183 277 483 N CALPA1 12-835-54 278501 N CALPA1 12-836-134 279 501 N CALPA1 12-836-237 280 500 N CALPA112-836-238 281 476 N CALPA1 12-836-257 282 498 N CALPA1 12-836-275 283501 N CALPA1 12-838-179 284 501 N CALPA1 12-839-397 285 501 N CALPA112-840-47 286 501 N CALPA1 12-840-77 287 501 N CALPA1 12-841-445 288 445N CALPA1 12-842-215 289 501 N CALPA1 12-842-447 290 499 N CALPA112-844-167 291 501 N CALPA1 12-845-364 292 501 N CALPA1 12-846-209 293501 N CALPA1 12-847-123 294 501 N CALPA1 12-849-242 295 501 N CYP2J210-336-58 296 501 N CYP2J2 10-336-137 297 501 N CYP2J2 10-336-232 298501 N CYP2J2 12-102-104 299 379 N CYP2J2 12-102-111 300 386 N CYP2J212-102-275 301 501 N CYP2J2 12-103-202 302 501 Y C 14.3 CYP2J212-103-214 303 501 N CYP2J2 12-104-351 304 501 Y T 27.4 CYP2J212-105-435 305 439 N CYP2J2 12-109-149 306 278 Y A 8.51 CYP2J212-109-197 307 326 N CYP2J2 12-109-209 308 338 N CYP2J2 12-109-284 309413 N CYP2J2 12-113-276 310 501 Y G 31.2 CYP2J2 12-115-57 311 501 Y G8.87 CYP2J2 12-119-26 312 501 Y G 29.8 COX1 12-347-308 313 501 N COX112-354-334 314 501 Y C/C COX1 12-357-140 315 501 Y C 7.14 COX112-361-320 316 501 Y G 18.3 COX1 12-361-388 317 501 Y A 18.5 COX112-365-251 318 501 Y C 18.8 COX1 12-374-261 319 501 Y T 21.3 COX110-308-116 320 501 N COX1 10-311-274 321 501 N COX1 10-314-76 322 501 NCOX1 10-306-265 323 501 N COX2 10-52-386 324 386 N COX2 10-62-240 325240 Y C 12.23 COX2 10-65-276 326 276 Y COX2 10-67-42 327 42 N COX210-67-340 328 341 Y COX2 10-55-265 329 264 Y C 40.9 COX2 10-57-278 330278 Y COX2 10-59-176 331 176 Y COX2 10-60-114 332 114 N PGDS 10-27-176333 176 Y A 5.32 PGDS 10-28-242 334 242 Y PGDS 10-30-349 335 350 Y A/APGDS 10-181-42 336 42 Y C 30.2 PGDS 10-181-372 337 374 Y C 26.3 PGDS10-183-260 338 259 N PG15OH 10-475-163 339 501 N PG15OH 12-884-203 340501 Y T 29.7 PG15OH 10-479-266 341 501 N PG15OH 10-479-350 342 501 NPG15OH 10-479-394 343 501 N PG15OH 10-482-145 344 501 N PG15OH 12-854-64345 501 N PG15OH 12-854-472 346 501 N PG15OH 12-855-194 347 501 N PG15OH12-855-288 348 501 N PG15OH 12-855-423 349 501 N PG15OH 12-857-25 350476 N PG15OH 12-858-346 351 501 Y T 37.2 PG15OH 12-858-443 352 501 NPG15OH 12-860-388 353 501 N PG15OH 12-861-270 354 501 N PG15OH12-862-349 355 501 N PG15OH 12-862-365 356 501 N PG15OH 12-862-452 357501 N PG15OH 12-866-423 358 501 Y C 46.2 PG15OH 12-867-47 359 501 NPG15OH 12-868-181 360 501 N PG15OH 12-868-198 361 501 N PG15OH12-868-282 362 501 N PG15OH 12-869-128 363 501 N PG15OH 12-870-491 364501 N PG15OH 12-872-52 365 501 N PG15OH 12-872-293 366 501 N PG15OH12-873-185 367 501 N PG15OH 12-873-319 368 501 N PG15OH 12-875-248 369501 Y G 28.8 PG15OH 12-876-265 370 501 N PG15OH 12-876-280 371 501 NPG15OH 12-876-454 372 501 N PG15OH 12-877-59 373 501 N PG15OH 12-877-69374 501 N PG15OH 12-877-79 375 501 N PG15OH 12-878-153 376 501 N PG15OH12-878-419 377 501 N PG15OH 12-879-67 378 501 N PG15OH 12-879-439 379501 N PG15OH 12-881-210 380 501 N PG15OH 12-881-389 381 501 N PG15OH12-883-273 382 501 N PG15OH 12-885-196 383 501 N PG15OH 12-885-333 384501 N PG15OH 12-885-407 385 501 N PG15OH 12-885-410 386 501 N PG15OH12-886-195 387 501 Y A 21.1 PG15OH 12-886-348 388 501 N PG15OH12-887-201 389 501 N PG15OH 12-887-467 390 501 N PG15OH 12-888-98 391501 N PG15OH 12-888-203 392 501 Y G 38.3 PG15OH 12-888-315 393 501 NPG15OH 12-889-518 394 479 N PG15OH 12-894-266 395 501 N PG15OH12-895-391 396 501 Y C 34.6 PG15OH 12-896-140 397 501 N PG15OH12-897-115 398 501 N PG15OH 12-897-225 399 501 N PG15OH 12-898-49 400528 N CYP8 12-164-119 401 501 Y C 11.8 CYP8 12-168-84 402 501 Y T 20.1CYP8 12-168-365 403 501 N CYP8 12-170-299 404 501 Y T 6.52 CYP812-171-360 405 501 Y T 8.70 CYP8 12-173-59 406 501 Y G 26.0 CYP812-175-214 407 501 Y A 10.1 CYP8 12-177-183 408 501 Y G 25.4 CYP812-177-366 409 501 N TAX2 10-128-45 410 45 Y T/T TAX2 10-128-63 411 63 NTAX2 10-123-177 412 177 N TAX2 10-123-402 413 402 N TAX2 10-120-137 414136 Y A 1.60 TAX2 10-120-141 415 140 Y A 3.09 TAX2 10-179-39 416 39 NTAX2 10-180-65 417 65 Y C 44.7 TAX2 10-179-257 418 257 Y 15-LOA10-43-124 419 123 N 15-LOA 10-43-134 420 133 N 15-LOA 10-43-193 421 192N 15-LOA 10-43-195 422 194 N 15-LOA 10-43-233 423 232 N 15-LOA 10-43-138424 137 Y 15-LOA 10-46-372 425 369 Y T 2.43 15-LOA 10-46-36 426 35 N15-LOA 10-47-103 427 102 Y 15-LOA 10-47-125 428 124 Y T 5.68 15-LOA10-48-184 429 183 Y T 28.0 15-LOA 10-48-381 430 382 Y T 31.4 15-LOA10-49-33 431 33 Y T 14.3 15-LOA 10-39-148 432 150 Y G 14.5 15-LOA10-40-222 433 222 Y A 47.6 15-LOA 10-40-252 434 250 N 15-LOA 10-42-354435 354 Y 15-LOA 10-154-42 436 42 N 15-LOA 10-154-156 437 156 Y T 24.215-LOA 10-154-226 438 226 N 15-LOB 12-776-259 439 501 N 5-LO 10-384-109440 501 N 5-LO 12-296-388 441 501 Y G 37.6 5-LO 10-388-379 442 501 N5-LO 10-389-116 443 501 N 5-LO 10-389-349 444 501 N 5-LO 10-391-94 445501 N 5-LO 12-277-147 446 501 Y T 44.9 5-LO 12-278-413 447 501 Y A 33.95-LO 12-288-190 448 501 N 5-LO 12-289-35 449 501 N 5-LO 12-296-119 450501 N 5-LO 12-297-291 451 501 N 5-LO 12-298-105 452 501 N 5-LO12-300-126 453 501 N 5-LO 12-300-410 454 501 N 5-LO 12-301-379 455 501 N5-LO 12-302-264 456 501 N 5-LO 12-309-405 457 501 N 5-LO 12-310-105 458501 N 5-LO 12-314-453 459 501 Y A 18.8 5-LO 12-316-292 460 501 Y C 40.8LTA4H 10-281-314 461 501 N LTA4H 10-268-381 462 501 N LTA4H 12-54-297463 501 Y C 9.34 LTA4H 10-276-407 464 501 N LTA4H 12-44-50 465 501 Y A25.9 LTA4H 12-44-67 466 501 N LTA4H 12-45-145 467 501 N LTA4H 12-45-166468 501 N LTA4H 12-45-305 469 501 N LTA4H 12-46-92 470 501 Y G 31.9LTA4H 12-47-132 471 501 Y C 4.84 LTA4H 12-47-61 472 501 N LTA4H12-48-100 473 501 N LTA4H 12-48-323 474 501 N LTA4H 12-48-369 475 501 NLTA4H 12-48-37 476 501 N LTA4H 12-49-131 477 501 Y A 40.1 LTA4H 12-49-53478 501 N LTA4H 12-49-64 479 501 N LTA4H 12-51-234 480 501 Y A 43.3LTA4H 12-51-253 481 501 N LTA4H 12-51-370 482 501 N LTA4H 12-52-400 483501 N LTA4H 12-57-192 484 501 Y T 41.2 LTA4H 12-57-221 485 501 Y T 4.40LTA4H 12-57-510 486 501 N LTB4H2 10-1-139 487 139 Y G 36.3 LTB4H210-1-212 488 212 Y T 16.3 LTB4H2 10-1-241 489 241 Y A 5.84 LTB4H210-9-143 490 143 Y LTB4H2 10-9-185 491 185 Y T/T LTB4H2 10-9-264 492 264Y LTB4H2 10-11-22 493 22 N LTB4H2 10-13-152 494 152 Y T 20.8 LTB4H210-13-256 495 256 Y LTB4H2 10-13-282 496 282 Y C 25.0 LTB4H2 10-15-281497 281 N LTB4H2 10-17-142 498 142 Y C/C LTB4H2 10-18-302 499 302 NLTB4H2 10-23-331 500 331 N LTB4H2 10-25-152 501 152 Y LTB4H2 10-25-258502 258 N LTB4H2 10-3-103 503 103 Y T 47.7 LTB4H2 10-3-144 504 144 YLTB4H2 10-3-275 505 275 Y LTB4H2 10-5-227 506 227 Y A 28.1 LTB4H210-7-155 507 155 Y T 30.4 LTB4H2 10-7-383 508 381 N LTB4H2 10-7-98 50998 N LTB412OH 12-561-270 510 501 Y T 35.2 LTB412OH 12-563-87 511 501 Y C28.0 LTB412OH 12-564-64 512 501 Y T 36.0 LTB412OH 12-564-214 513 501 NLTB412OH 12-568-207 514 501 N LTB412OH 12-568-365 515 501 N LTB412OH12-568-367 516 501 N LTB412OH 12-571-337 517 501 Y G 17.9 LTB412OH12-573-378 518 501 Y A 6.91 LTB412OH 10-294-256 519 501 N LTB412OH10-294-304 520 501 N LTB412OH 10-295-201 521 501 N LTB412OH 10-296-80522 501 N LTB412OH 10-296-373 523 501 N LTB412OH 10-298-122 524 501 NLTB412OH 10-298-158 525 501 N LTB412OH 10-300-49 526 501 N LTB412OH10-300-185 527 501 N LTB4H3 10-10-328 528 327 Y A 12.5 LTB4H3 10-12-52529 52 N LTB4H3 10-14-46 530 46 Y T 39.3 LTB4H3 10-19-358 531 357 YLTB4H3 10-20-111 532 110 Y A 15.8 LTB4H3 10-20-274 533 273 Y A/A LTB4H310-24-90 534 90 Y C 19.2 LTB4H3 10-24-204 535 204 Y A 25.0 LTB4H310-24-221 536 221 N LTB4H3 10-24-234 537 234 Y A 36.1 LTB4H3 10-24-288538 288 N LTB4H3 10-24-311 539 311 N LTB4H3 10-26-289 540 289 N LTB4H310-8-39 541 39 Y LTB4H3 10-8-120 542 120 N LTB4H3 10-8-154 543 154 NLTB4H3 10-8-101 544 101 Y LTB4H3 10-8-86 545 86 Y LTB4H3 10-8-92 546 92N LTB4H3 10-8-94 547 94 N LTB4R 12-61-472 548 501 N LTB4R 12-63-402 549416 N LTB4R 12-63-74 550 88 N LTB4R 12-64-271 551 287 Y C 28.6 LTB4R12-65-98 552 439 N LTB4R 12-70-147 553 501 Y C 11.5 LTB4R 12-70-397 554501 Y T 39.7 LTB4R 12-71-320 555 501 Y A 4.49 LTB4R 12-73-150 556 501 NLTB4R 12-73-49 557 501 Y A 43.3 LTB4R 12-73-56 558 501 N LTB4R 12-74-38559 501 Y C 44.1 LTB4R 12-76-238 560 501 Y T 20.6 LTB4R 12-77-217 561501 N LTB4R 12-77-478 562 501 Y A 4.40 LTB4R 12-80-114 563 501 N LTB4R12-80-233 564 501 Y C 4.55 LTB4R 12-82-250 565 250 N LTC4 10-176-85 56685 Y T 0.54 LTC4 10-176-51 567 51 N LTC4 10-176-207 568 207 N LTC410-176-397 569 397 Y A 1.63 LTC4 10-177-219 570 219 Y C 29.0 12-LO12-214-85 571 85 N 12-LO 12-215-272 572 271 N 12-LO 12-221-163 573 163 N12-LO 12-225-82 574 82 N cPLA₂ 10-234-179 575 214 Y Deletion AA 32.6cPLA₂ 10-235-272 576 491 N ANX1 10-251-342 577 498 N ANX2 10-395-367 578497 N ANX2 12-730-58 579 498 N ANX2 12-735-208 580 412 Y Deletion 21.5ANX2 12-739-22 581 498 Y Insertion G 23.4 ANX3 12-540-363 582 498 N ANX312-550-206 583 497 N CAL2 12-207-410 584 409 N CAL3 10-171-254 585 255 NCALPA1 12-94-110 586 498 Y Deletion 32.5 AATT CALPA1 12-834-290 587 498N COX2 10-55-115 588 114 Y Deletion 3.01 TTATA PG15OH 12-857-122 589 498N PG15OH 12-872-175 590 498 N PG15OH 12-882-40 591 498 N PG15OH12-888-234 592 498 N 5-LO 12-278-353 593 499 N 5-LO 12-283-386 594 498 NLTA4H 12-44-181 595 458 N ANX3 10-370-132 596 501 N ANX3 10-370-254 597501 N 15PGDHB 10-485-256 598 501 N 15PGDHB 10-485-257 599 501 N 15PGDHB10-474-320 600 501 N 5LO 10-387-371 601 501 N LTB412OH 12-570-239 602501 N LTB412OH 12-570-344 603 501 N LTB412OH 12-570-393 604 501 NLTB412OH 12-570-421 605 501 N LTB412OH 12-570-62 606 502 N LTB4H310-4-144 607 141 N LTB4H3 10-4-161 608 158 N LTB4H3 10-4-270 609 267 NLTB4H3 10-4-340 610 337 N LTB4H3 10-4-369 611 366 N LTB4H3 10-4-420 612417 N LTB4H2 10-13-396 613 396 N 12-LO 10-509-284 614 501 N 12-LO10-509-295 615 501 N 12-LO 10-339-124 616 501 N 12-LO 10-340-112 617 501N 12-LO 10-340-130 618 501 N 12-LO 10-340-238 619 501 N 12-LO 10-342-301620 501 N 12-LO 10-342-373 621 501 N 12-LO 10-343-231 622 501 N 12-LO10-343-278 623 501 N 12-LO 10-346-141 624 501 N G/G 12-LO 10-346-23 625500 N 12-LO 10-346-263 626 501 N 12-LO 10-346-305 627 501 N 12-LO10-349-216 628 501 N 12-LO 10-350-332 629 501 N 12-LO 10-350-72 630 501N 12-LO 10-507-170 631 501 N 12-LO 10-507-321 632 501 N 12-LO 10-507-353633 501 N 12-LO 10-507-364 634 501 N 12-LO 10-507-405 635 501 N 12-LO10-508-191 636 501 N 12-LO 10-508-245 637 501 N 12-LO 10-510-173 638 501N 12-LO 10-511-337 639 501 N 12-LO 10-512-36 640 501 Y C 39.4 12-LO10-511-62 641 501 N 12-LO 10-512-318 642 501 N 12-LO 10-513-250 643 501N 12-LO 10-513-262 644 501 N 12-LO 10-513-352 645 501 N 12-LO 10-513-365646 501 N FLAP 10-517-100 647 501 N FLAP 10-518-125 648 501 N FLAP10-518-194 649 501 N FLAP 10-522-71 650 501 N

[0756] TABLE 7B List of all of the eicosanoid-related biallelic markers(47mers) BIALLELIC GENOTYPING MARKER VALIDATION LEAST COMMON BIALLELICSEQ ID POSITION IN MICRO- ALLELE GENE MARKER ID NO. SEQ ID NO.SEQUENCING FREQUENCY % FLAP 10-253-118 655 24 N FLAP 10-253-298 656 24 YG 4.57 FLAP 10-253-315 657 24 N FLAP 10-499-155 658 24 N FLAP 10-520-256659 24 N T 40.8 FLAP 10-500-258 660 24 N FLAP 10-500-410 661 24 N FLAP10-503-159 662 24 N FLAP 10-504-172 663 24 N FLAP 10-504-243 664 24 NFLAP 10-204-326 665 24 Y A 6.63 FLAP 10-32-357 666 24 Y A 33.5 FLAP10-33-175 667 24 Y T 2.30 FLAP 10-33-211 668 24 N FLAP 10-33-234 669 24Y A 44.0 FLAP 10-33-270 670 24 Y G/G FLAP 10-33-327 671 24 Y C 24.5 FLAP10-34-290 672 24 N FLAP 10-35-358 673 24 Y C 31.3 FLAP 10-35-390 674 24Y T 23.0 FLAP 10-36-164 675 24 Y G/G FLAP 10-498-192 676 24 N FLAP12-628-306 677 24 Y T 10.3 FLAP 12-628-311 678 24 N FLAP 12-629-241 67924 Y C 28.3 12-LO 12-206-366 680 24 Y C 38.2 12-LO 10-343-339 681 24 N12-LO 10-347-74 682 24 N 12-LO 10-347-111 683 24 N G/G 12-LO 10-347-165684 24 N C/C 12-LO 10-347-203 685 24 Y G 41.6 12-LO 10-347-220 686 24 YA 40.5 12-LO 10-347-271 687 24 N 12-LO 10-347-348 688 24 N 12-LO10-348-391 689 24 N 12-LO 10-349-47 690 24 N 12-LO 10-349-97 691 24 Y G39.6 12-LO 10-349-142 692 24 N C/C 12-LO 10-349-224 693 24 Y T 39.612-LO 10-349-368 694 24 N 12-LO 10-339-32 695 24 N 12-LO 10-341-116 69624 Y A 10.8 12-LO 10-341-319 697 24 N 12-LO 12-196-119 698 24 Y C 29.112-LO 12-197-244 699 24 Y C 32.8 12-LO 12-198-128 700 24 N 12-LO12-206-81 701 24 N 12-LO 12-208-35 702 24 Y A 42.3 12-LO 12-214-129 70324 Y C 38.7 12-LO 12-214-151 704 24 N 12-LO 12-214-360 705 24 N 12-LO12-215-467 706 24 N 12-LO 12-216-421 707 24 Y A 36.0 12-LO 12-219-230708 24 Y G 32.1 12-LO 12-219-256 709 24 N 12-LO 12-220-48 710 24 N 12-LO12-221-302 711 24 N 12-LO 12-223-179 712 24 N 12-LO 12-223-207 713 24 YC 38.4 12-LO 12-225-541 714 24 Y C 37.4 12-LO 12-226-167 715 24 Y G 41.212-LO 12-226-458 716 24 N 12-LO 12-229-332 717 24 N 12-LO 12-229-351 71824 N 12-LO 12-230-364 719 24 N 12-LO 12-231-100 720 24 N 12-LO12-231-148 721 24 N 12-LO 12-231-266 722 24 N cPLA₂ 10-231-23 723 24 Y A8.79 cPLA₂ 10-233-386 724 24 Y G 28.3 cPLA₂ 10-239-368 726 24 N cPLA₂10-223-30 727 24 Y G 22.5 cPLA₂ 10-223-72 728 24 N cPLA₂ 10-223-130 72924 N cPLA₂ 10-223-262 730 24 N cPLA₂ 10-223-392 731 24 N cPLA₂10-224-341 732 24 N cPLA₂ 10-227-282 733 24 Y G 3.93 ANX1 10-240-241 73424 N ANX1 10-249-185 735 24 N ANX1 10-251-128 736 24 N ANX1 10-252-209737 24 N ANX1 12-387-32 738 24 Y G 33.9 ANX1 10-242-316 739 24 N ANX110-245-412 740 24 N ANX1 12-378-171 741 24 N ANX1 12-378-228 742 24 NANX1 12-378-450 743 24 N ANX1 12-379-65 744 24 N ANX1 12-382-204 745 24Y G 50.0 ANX1 12-383-117 746 24 N ANX1 12-383-170 747 24 N ANX112-383-268 748 24 N ANX1 12-384-336 749 24 N ANX1 12-384-451 750 24 NANX1 12-385-123 751 24 N ANX1 12-385-427 752 24 N ANX1 12-386-155 753 24Y G 8.15 ANX1 12-386-24 754 24 N ANX1 12-387-177 755 24 Y T 33.5 ANX112-389-431 756 24 N ANX1 12-391-366 757 24 N ANX1 12-394-85 758 24 NANX1 12-395-382 759 24 N ANX1 12-400-217 760 24 Y G 27.2 ANX1 12-400-280761 24 N ANX1 12-401-378 762 24 N ANX1 12-402-126 763 24 N ANX112-404-265 764 24 N ANX1 12-406-52 765 24 N ANX1 12-406-409 766 24 NANX1 12-407-217 767 24 N ANX1 12-407-399 768 24 N ANX1 12-408-355 769 24Y G 2.69 ANX1 12-409-221 770 24 N ANX1 12-410-301 771 24 N ANX210-395-101 772 24 N ANX2 10-395-124 773 24 N ANX2 10-395-155 774 24 NANX2 10-395-294 775 24 N ANX2 10-396-100 776 24 N ANX2 10-397-201 777 24N ANX2 10-399-178 778 24 N ANX2 10-400-369 779 24 N ANX2 10-392-20 78024 N ANX2 10-392-103 781 24 N ANX2 10-392-324 782 24 N ANX2 10-393-27783 24 N ANX2 10-393-324 784 24 N ANX2 12-727-237 785 24 N ANX212-728-224 786 24 N ANX2 12-730-142 787 24 N ANX2 12-730-193 788 24 NANX2 12-731-60 789 24 N ANX2 12-731-119 790 24 N ANX2 12-731-137 791 24N ANX2 12-731-146 792 24 N ANX2 12-731-398 793 24 N ANX2 12-732-113 79424 N ANX2 12-732-164 795 24 N ANX2 12-732-165 796 24 Y C 27.4 ANX212-732-445 797 24 N ANX2 12-734-201 798 24 N ANX2 12-735-42 799 24 NANX2 12-736-363 800 24 N ANX2 12-737-69 801 24 Y A 36.8 ANX2 12-737-296802 24 N ANX2 12-738-429 803 24 Y T 35.5 ANX2 12-740-112 804 24 Y G 37.6ANX2 12-740-118 805 24 N ANX2 12-741-265 806 24 N ANX2 12-741-327 807 24N ANX2 12-741-376 808 24 N ANX2 12-745-30 809 24 N ANX2 12-745-75 810 24N ANX2 12-745-343 811 24 N ANX2 12-745-350 812 24 N ANX2 12-746-320 81324 N ANX2 12-747-181 814 24 N ANX2 12-747-302 815 24 N ANX2 12-749-240816 24 N ANX2 12-749-255 817 24 N ANX2 12-752-37 818 24 N ANX2 12-752-85819 24 N ANX2 12-752-196 820 24 N ANX2 12-752-484 821 24 N ANX212-753-139 822 24 N ANX2 12-753-376 823 24 N ANX2 12-754-172 824 24 NANX2 12-754-218 825 24 N ANX2 12-754-328 826 24 N ANX2 12-754-396 827 24N ANX2 12-755-280 828 24 N ANX2 12-757-384 829 24 N ANX2 12-758-257 83024 N ANX2 12-758-374 831 24 N ANX2 12-758-424 832 24 N ANX2 12-761-23833 24 N ANX2 12-761-178 834 24 N ANX2 12-764-329 835 24 N ANX212-764-377 836 24 N ANX2 12-765-168 837 24 N ANX2 12-765-504 838 24 NANX3 10-372-279 839 24 N ANX3 10-375-136 840 24 N ANX3 10-376-281 841 24N ANX3 10-369-392 842 24 N ANX3 10-371-257 843 24 N ANX3 12-513-389 84424 N ANX3 12-513-494 845 24 N ANX3 12-515-394 846 24 N ANX3 12-516-97847 24 Y T 37.2 ANX3 12-520-287 848 24 N ANX3 12-520-323 849 24 Y A 21.5ANX3 12-523-179 850 24 Y A 29.9 ANX3 12-523-270 851 24 N ANX3 12-527-367852 24 Y T 18.9 ANX3 12-529-376 853 24 N ANX3 12-529-489 854 24 N ANX312-530-134 855 24 Y T 39.3 ANX3 12-530-393 856 24 N ANX3 12-531-173 85724 Y C 37.6 ANX3 12-539-441 858 24 N ANX3 12-543-78 859 24 N ANX312-543-79 860 24 N ANX3 12-546-235 861 24 N ANX3 12-549-287 862 24 NANX3 12-550-287 863 24 N ANX3 12-552-175 864 24 N ANX3 12-554-330 865 24N ANX3 12-556-312 866 24 N ANX3 12-556-443 867 24 N ANX3 12-558-205 86824 N ANX3 12-558-238 869 24 N ANX3 12-558-305 870 24 N ANX3 12-769-39871 24 N ANX3 12-769-430 872 24 N ANX3 12-770-73 873 24 N ANX312-772-200 874 24 N ANX3 12-772-254 875 24 N CAL1 10-87-73 876 24 N CAL110-87-74 877 24 N CAL1 10-87-80 878 24 N CAL1 10-87-140 879 24 N CAL110-88-81 880 24 Y C 44.7 CAL1 10-89-41 881 24 N CAL1 10-90-35 882 24 Y A1.14 CAL1 10-91-274 883 24 N CAL1 10-93-133 884 24 N CAL1 10-94-197 88524 Y G/G CAL1 10-94-198 886 24 N CAL1 10-166-362 887 24 N CAL210-207-386 888 24 Y C/C CAL2 10-207-409 889 24 Y G 9.04 CAL2 10-118-307890 24 Y A 0.27 CAL2 10-173-247 891 24 N CAL2 10-173-294 892 24 Y G 2.87CAL2 10-173-347 893 24 Y C/C CAL3 10-103-104 894 24 N CAL3 10-103-323895 24 Y T 22.3 CAL3 10-103-402 896 24 N CAL3 10-106-98 897 24 N CAL310-106-288 898 24 Y CAL3 10-106-378 899 24 Y CAL3 10-168-160 900 24 Y T42.1 CAL3 10-168-206 901 24 Y CAL3 10-168-284 902 24 N CAL3 10-169-318903 24 N CALPA1 12-86-79 904 24 Y C 37.4 CALPA1 12-88-393 905 24 NCALPA1 12-89-369 906 24 Y G 36.3 CALPA1 12-89-91 907 24 N CALPA112-94-210 908 24 N CALPA1 12-94-516 909 24 N CALPA1 12-96-64 910 24 Y T8.52 CALPA1 12-97-83 911 24 N CALPA1 12-99-296 912 24 Y T 6.45 CALPA112-100-266 913 24 Y G 32.2 CALPA1 12-811-174 914 24 N CALPA1 12-815-94915 24 N CALPA1 12-815-383 916 24 N CALPA1 12-815-384 917 24 N CALPA112-815-391 918 24 N CALPA1 12-817-214 919 24 N CALPA1 12-817-355 920 24N CALPA1 12-819-437 921 24 N CALPA1 12-821-62 922 24 N CALPA1 12-821-483923 24 N CALPA1 12-825-173 924 24 N CALPA1 12-826-312 925 24 N CALPA112-831-59 926 24 N CALPA1 12-833-264 927 24 N CALPA1 12-833-279 928 24 NCALPA1 12-833-280 929 24 N CALPA1 12-833-373 930 24 N CALPA1 12-834-183931 24 N CALPA1 12-835-54 932 24 N CALPA1 12-836-134 933 24 N CALPA112-836-237 934 24 N CALPA1 12-836-238 935 24 N CALPA1 12-836-257 936 24N CALPA1 12-836-275 937 24 N CALPA1 12-838-179 938 24 N CALPA112-839-397 939 24 N CALPA1 12-840-47 940 24 N CALPA1 12-840-77 941 24 NCALPA1 12-841-445 942 24 N CALPA1 12-842-215 943 24 N CALPA1 12-842-447944 24 N CALPA1 12-844-167 945 24 N CALPA1 12-845-364 946 24 N CALPA112-846-209 947 24 N CALPA1 12-847-123 948 24 N CALPA1 12-849-242 949 24N CYP2J2 10-336-58 950 24 N CYP2J2 10-336-137 951 24 N CYP2J2 10-336-232952 24 N CYP2J2 12-102-104 953 24 N CYP2J2 12-102-111 954 24 N CYP2J212-102-275 955 24 N CYP2J2 12-103-202 956 24 Y C 14.3 CYP2J2 12-103-214957 24 N CYP2J2 12-104-351 958 24 Y T 27.4 CYP2J2 12-105-435 959 24 NCYP2J2 12-109-149 960 24 Y A 8.51 CYP2J2 12-109-197 961 24 N CYP2J212-109-209 962 24 N CYP2J2 12-109-284 963 24 N CYP2J2 12-113-276 964 24Y G 31.2 CYP2J2 12-115-57 965 24 Y G 8.87 CYP2J2 12-119-26 966 24 Y G29.8 COX1 12-347-308 967 24 N COX1 12-354-334 968 24 Y C/C COX112-357-140 969 24 Y C 7.14 COX1 12-361-320 970 24 Y G 18.3 COX112-361-388 971 24 Y A 18.5 COX1 12-365-251 972 24 Y C 18.8 COX112-374-261 973 24 Y T 21.3 COX1 10-308-116 974 24 N COX1 10-311-274 97524 N COX1 10-314-76 976 24 N COX1 10-306-265 977 24 N COX2 10-52-386 97824 N COX2 10-62-240 979 24 Y C 12.23 COX2 10-65-276 980 24 Y COX210-67-42 981 24 N COX2 10-67-340 982 24 Y COX2 10-55-265 983 24 Y C 40.9COX2 10-57-278 984 24 Y COX2 10-59-176 985 24 Y COX2 10-60-114 986 24 NPGDS 10-27-176 987 24 Y A 5.32 PGDS 10-28-242 988 24 Y PGDS 10-30-349989 24 Y A/A PGDS 10-181-42 990 24 Y C 30.2 PGDS 10-181-372 991 24 Y C26.3 PGDS 10-183-260 992 24 N PG15OH 10-475-163 993 24 N PG15OH12-884-203 994 24 Y T 29.7 PG15OH 10-479-266 995 24 N PG15OH 10-479-350996 24 N PG15OH 10-479-394 997 24 N PG15OH 10-482-145 998 24 N PG15OH12-854-64 999 24 N PG15OH 12-854-472 1000 24 N PG15OH 12-855-194 1001 24N PG15OH 12-855-288 1002 24 N PG15OH 12-855-423 1003 24 N PG15OH12-857-25 1004 24 N PG15OH 12-858-346 1005 24 Y T 37.2 PG15OH 12-858-4431006 24 N PG15OH 12-860-388 1007 24 N PG15OH 12-861-270 1008 24 N PG15OH12-862-349 1009 24 N PG15OH 12-862-365 1010 24 N PG15OH 12-862-452 101124 N PG15OH 12-866-423 1012 24 Y C 46.2 PG15OH 12-867-47 1013 24 NPG15OH 12-868-181 1014 24 N PG15OH 12-868-198 1015 24 N PG15OH12-868-282 1016 24 N PG15OH 12-869-128 1017 24 N PG15OH 12-870-491 101824 N PG15OH 12-872-52 1019 24 N PG15OH 12-872-293 1020 24 N PG15OH12-873-185 1021 24 N PG15OH 12-873-319 1022 24 N PG15OH 12-875-248 102324 Y G 28.8 PG15OH 12-876-265 1024 24 N PG15OH 12-876-280 1025 24 NPG15OH 12-876-454 1026 24 N PG15OH 12-877-59 1027 24 N PG15OH 12-877-691028 24 N PG15OH 12-877-79 1029 24 N PG15OH 12-878-153 1030 24 N PG15OH12-878-419 1031 24 N PG15OH 12-879-67 1032 24 N PG15OH 12-879-439 103324 N PG15OH 12-881-210 1034 24 N PG15OH 12-881-389 1035 24 N PG15OH12-883-273 1036 24 N PG15OH 12-885-196 1037 24 N PG15OH 12-885-333 103824 N PG15OH 12-885-407 1039 24 N PG15OH 12-885-410 1040 24 N PG15OH12-886-195 1041 24 Y A 21.1 PG15OH 12-886-348 1042 24 N PG15OH12-887-201 1043 24 N PG15OH 12-887-467 1044 24 N PG15OH 12-888-98 104524 N PG15OH 12-888-203 1046 24 Y G 38.3 PG15OH 12-888-315 1047 24 NPG15OH 12-889-518 1048 24 N PG15OH 12-894-266 1049 24 N PG15OH12-895-391 1050 24 Y C 34.6 PG15OH 12-896-140 1051 24 N PG15OH12-897-115 1052 24 N PG15OH 12-897-225 1053 24 N PG15OH 12-898-49 105424 N CYP8 12-164-119 1055 24 Y C 11.8 CYP8 12-168-84 1056 24 Y T 20.1CYP8 12-168-365 1057 24 N CYP8 12-170-299 1058 24 Y T 6.52 CYP812-171-360 1059 24 Y T 8.70 CYP8 12-173-59 1060 24 Y G 26.0 CYP812-175-214 1061 24 Y A 10.1 CYP8 12-177-183 1062 24 Y G 25.4 CYP812-177-366 1063 24 N TAX2 10-128-45 1064 24 Y T/T TAX2 10-128-63 1065 24N TAX2 10-123-177 1066 24 N TAX2 10-123-402 1067 24 N TAX2 10-120-1371068 24 Y A 1.60 TAX2 10-120-141 1069 24 Y A 3.09 TAX2 10-179-39 1070 24N TAX2 10-180-65 1071 24 Y C 44.7 TAX2 10-179-257 1072 24 Y 15-LOA10-43-124 1073 24 N 15-LOA 10-43-134 1074 24 N 15-LOA 10-43-193 1075 24N 15-LOA 10-43-195 1076 24 N 15-LOA 10-43-233 1077 24 N 15-LOA 10-43-1381078 24 Y 15-LOA 10-46-372 1079 24 Y T 2.43 15-LOA 10-46-36 1080 24 N15-LOA 10-47-103 1081 24 Y 15-LOA 10-47-125 1082 24 Y T 5.68 15-LOA10-48-184 1083 24 Y T 28.0 15-LOA 10-48-381 1084 24 Y T 31.4 15-LOA10-49-33 1085 24 Y T 14.3 15-LOA 10-39-148 1086 24 Y G 14.5 15-LOA10-40-222 1087 24 Y A 47.6 15-LOA 10-40-252 1088 24 N 15-LOA 10-42-3541089 24 Y 15-LOA 10-154-42 1090 24 N 15-LOA 10-154-156 1091 24 Y T 24.215-LOA 10-154-226 1092 24 N 15-LOB 12-776-259 1093 24 N 5-LO 10-384-1091094 24 N 5-LO 12-296-388 1095 24 Y G 37.6 5-LO 10-388-379 1096 24 N5-LO 10-389-116 1097 24 N 5-LO 10-389-349 1098 24 N 5-LO 10-391-94 109924 N 5-LO 12-277-147 1100 24 Y T 44.9 5-LO 12-278-413 1101 24 Y A 33.95-LO 12-288-190 1102 24 N 5-LO 12-289-35 1103 24 N 5-LO 12-296-119 110424 N 5-LO 12-297-291 1105 24 N 5-LO 12-298-105 1106 24 N 5-LO 12-300-1261107 24 N 5-LO 12-300-410 1108 24 N 5-LO 12-301-379 1109 24 N 5-LO12-302-264 1110 24 N 5-LO 12-309-405 1111 24 N 5-LO 12-310-105 1112 24 N5-LO 12-314-453 1113 24 Y A 18.8 5-LO 12-316-292 1114 24 Y C 40.8 LTA4H10-281-314 1115 24 N LTA4H 10-268-381 1116 24 N LTA4H 12-54-297 1117 24Y C 9.34 LTA4H 10-276-407 1118 24 N LTA4H 12-44-50 1119 24 Y A 25.9LTA4H 12-44-67 1120 24 N LTA4H 12-45-145 1121 24 N LTA4H 12-45-166 112224 N LTA4H 12-45-305 1123 24 N LTA4H 12-46-92 1124 24 Y G 31.9 LTA4H12-47-132 1125 24 Y C 4.84 LTA4H 12-47-61 1126 24 N LTA4H 12-48-100 112724 N LTA4H 12-48-323 1128 24 N LTA4H 12-48-369 1129 24 N LTA4H 12-48-371130 24 N LTA4H 12-49-131 1131 24 Y A 40.1 LTA4H 12-49-53 1132 24 NLTA4H 12-49-64 1133 24 N LTA4H 12-51-234 1134 24 Y A 43.3 LTA4H12-51-253 1135 24 N LTA4H 12-51-370 1136 24 N LTA4H 12-52-400 1137 24 NLTA4H 12-57-192 1138 24 Y T 41.2 LTA4H 12-57-221 1139 24 Y T 4.40 LTA4H12-57-510 1140 24 N LTB4H2 10-1-139 1141 24 Y G 36.3 LTB4H2 10-1-2121142 24 Y T 16.3 LTB4H2 10-1-241 1143 24 Y A 5.84 LTB4H2 10-9-143 114424 Y LTB4H2 10-9-185 1145 24 Y T/T LTB4H2 10-9-264 1146 24 Y LTB4H210-11-22 1147 24 N LTB4H2 10-13-152 1148 24 Y T 20.8 LTB4H2 10-13-2561149 24 Y LTB4H2 10-13-282 1150 24 Y C 25.0 LTB4H2 10-15-281 1151 24 NLTB4H2 10-17-142 1152 24 Y C/C LTB4H2 10-18-302 1153 24 N LTB4H210-23-331 1154 24 N LTB4H2 10-25-152 1155 24 Y LTB4H2 10-25-258 1156 24N LTB4H2 10-3-103 1157 24 Y T 47.7 LTB4H2 10-3-144 1158 24 Y LTB4H210-3-275 1159 24 Y LTB4H2 10-5-227 1160 24 Y A 28.1 LTB4H2 10-7-155 116124 Y T 30.4 LTB4H2 10-7-383 1162 24 N LTB4H2 10-7-98 1163 24 N LTB412OH12-561-270 1164 24 Y T 35.2 LTB412OH 12-563-87 1165 24 Y C 28.0 LTB412OH12-564-64 1166 24 Y T 36.0 LTB412OH 12-564-214 1167 24 N LTB412OH12-568-207 1168 24 N LTB412OH 12-568-365 1169 24 N LTB412OH 12-568-3671170 24 N LTB412OH 12-571-337 1171 24 Y G 17.9 LTB412OH 12-573-378 117224 Y A 6.91 LTB412OH 10-294-256 1173 24 N LTB412OH 10-294-304 1174 24 NLTB412OH 10-295-201 1175 24 N LTB412OH 10-296-80 1176 24 N LTB412OH10-296-373 1177 24 N LTB412OH 10-298-122 1178 24 N LTB412OH 10-298-1581179 24 N LTB412OH 10-300-49 1180 24 N LTB412OH 10-300-185 1181 24 NLTB4H3 10-10-328 1182 24 Y A 12.5 LTB4H3 10-12-52 1183 24 N LTB4H310-14-46 1184 24 Y T 39.3 LTB4H3 10-19-358 1185 24 Y LTB4H3 10-20-1111186 24 Y A 15.8 LTB4H3 10-20-274 1187 24 Y A/A LTB4H3 10-24-90 1188 24Y C 19.2 LTB4H3 10-24-204 1189 24 Y A 25.0 LTB4H3 10-24-221 1190 24 NLTB4H3 10-24-234 1191 24 Y A 36.1 LTB4H3 10-24-288 1192 24 N LTB4H310-24-311 1193 24 N LTB4H3 10-26-289 1194 24 N LTB4H3 10-8-39 1195 24 YLTB4H3 10-8-120 1196 24 N LTB4H3 10-8-154 1197 24 N LTB4H3 10-8-101 119824 Y LTB4H3 10-8-86 1199 24 Y LTB4H3 10-8-92 1200 24 N LTB4H3 10-8-941201 24 N LTB4R 12-61-472 1202 24 N LTB4R 12-63-402 1203 24 N LTB4R12-63-74 1204 24 N LTB4R 12-64-271 1205 24 Y C 28.6 LTB4R 12-65-98 120624 N LTB4R 12-70-147 1207 24 Y C 11.5 LTB4R 12-70-397 1208 24 Y T 39.7LTB4R 12-71-320 1209 24 Y A 4.49 LTB4R 12-73-150 1210 24 N LTB4R12-73-49 1211 24 Y A 43.3 LTB4R 12-73-56 1212 24 N LTB4R 12-74-38 121324 Y C 44.1 LTB4R 12-76-238 1214 24 Y T 20.6 LTB4R 12-77-217 1215 24 NLTB4R 12-77-478 1216 24 Y A 4.40 LTB4R 12-80-114 1217 24 N LTB4R12-80-233 1218 24 Y C 4.55 LTB4R 12-82-250 1219 24 N LTC4 10-176-85 122024 Y T 0.54 LTC4 10-176-51 1221 24 N LTC4 10-176-207 1222 24 N LTC410-176-397 1223 24 Y A 1.63 LTC4 10-177-219 1224 24 Y C 29.0 12-LO12-214-85 1225 24 N 12-LO 12-215-272 1226 24 N 12-LO 12-221-163 1227 24N 12-LO 12-225-82 1228 24 N cPLA₂ 10-234-179 1229 24 Y Deletion 32.6 AAcPLA₂ 10-235-272 1230 24 N ANX1 10-251-342 1231 24 N ANX2 10-395-3671232 24 N ANX2 12-730-58 1233 24 N ANX2 12-735-208 1234 24 Y Deletion21.5 ANX2 12-739-22 1235 24 Y Insertion 23.4 G ANX3 12-540-363 1236 24 NANX3 12-550-206 1237 24 N CAL2 12-207-410 1238 24 N CAL3 10-171-254 123924 N CALPA1 12-94-110 1240 24 Y Deletion 32.5 AATT CALPA1 12-834-2901241 24 N COX2 10-55-115 1242 24 Y Deletion 3.01 TTATA PG15OH 12-857-1221243 24 N PG15OH 12-872-175 1244 24 N PG15OH 12-882-40 1245 24 N PG15OH12-888-234 1246 24 N 5-LO 12-278-353 1247 24 N 5-LO 12-283-386 1248 24 NLTA4H 12-44-181 1249 24 N ANX3 10-370-132 1250 24 N ANX3 10-370-254 125124 N 15PGDHB 10-485-256 1252 24 N 15PGDHB 10-485-257 1253 24 N 15PGDHB10-474-320 1254 24 N 5LO 10-387-371 1255 24 N LTB412OH 12-570-239 125624 N LTB412OH 12-570-344 1257 24 N LTB412OH 12-570-393 1258 24 NLTB412OH 12-570-421 1259 24 N LTB412OH 12-570-62 1260 24 N LTB4H310-4-144 1261 24 N LTB4H3 10-4-161 1262 24 N LTB4H3 10-4-270 1263 24 NLTB4H3 10-4-340 1264 24 N LTB4H3 10-4-369 1265 24 N LTB4H3 10-4-420 126624 N LTB4H2 10-13-396 1267 24 N 12-LO 10-509-284 1268 24 N 12-LO10-509-295 1269 24 N 12-LO 10-339-124 1270 24 N 12-LO 10-340-112 1271 24N 12-LO 10-340-130 1272 24 N 12-LO 10-340-238 1273 24 N 12-LO 10-342-3011274 24 N 12-LO 10-342-373 1275 24 N 12-LO 10-343-231 1276 24 N 12-LO10-343-278 1277 24 N 12-LO 10-346-141 1278 24 N G/G 12-LO 10-346-23 127924 N 12-LO 10-346-263 1280 24 N 12-LO 10-346-305 1281 24 N 12-LO10-349-216 1282 24 N 12-LO 10-350-332 1283 24 N 12-LO 10-350-72 1284 24N 12-LO 10-507-170 1285 24 N 12-LO 10-507-321 1286 24 N 12-LO 10-507-3531287 24 N 12-LO 10-507-364 1288 24 N 12-LO 10-507-405 1289 24 N 12-LO10-508-191 1290 24 N 12-LO 10-508-245 1291 24 N 12-LO 10-510-173 1292 24N 12-LO 10-511-337 1293 24 N 12-LO 10-512-36 1294 24 Y C 39.4 12-LO10-511-62 1295 24 N 12-LO 10-512-318 1296 24 N 12-LO 10-513-250 1297 24N 12-LO 10-513-262 1298 24 N 12-LO 10-513-352 1299 24 N 12-LO 10-513-3651300 24 N FLAP 10-517-100 1301 24 N FLAP 10-518-125 1302 24 N FLAP10-518-194 1303 24 N FLAP 10-522-71 1304 24 N

[0757] TABLE 8 SEQ ID BIALLELIC 1^(ST) 2^(ND) POSITION RANGE OF NO.MARKER ID ALLELE ALLELE PREFERRED SEQUENCE 1 10-253-118 A G [1-955] 210-253-298 G C [1-840] 3 10-253-315 C T [1-823] 4 10-499-155 A G[1-556], [898-955] 5 10-520-256 C T [1-384], [726-955] 6 10-500-258 G T[1-311], [653-955] 7 10-500-410 A G [1-160], [502-955] 8 10-503-159 G T[143-160], [388-408], [447-955] 9 10-504-172 A T [1-85], [124-792] 1010-504-243 A C [1-15], [54-722] 19 10-35-358 G C [555-842] 23 12-628-306G A [1-868], [904-955] 24 12-628-311 T C [1-873], [909-955] 2512-629-241 G C [1-17], [247-658], [705-787], [882-955] 27 10-343-339 G T[487-506], [733-904] 28 10-347-74 A G [1-134], [240-487], [784-956] 3510-348-391 A G [351-552], [682-776] 40 10-349-368 C T [416-525] 4412-196-119 C T [1-469] 45 12-197-244 C T [153-206] 48 12-208-35 A T[1-346], [453-507] 52 12-215-467 G T [1-161], [254-499] 53 12-216-421 AG [1-486] 54 12-219-230 A G [1-485] 55 12-219-256 C T [1-485] 5612-220-48 G A [1-577], [883-956] 57 12-221-302 A C [1-64], [265-286] 5812-223-179 A G [1-468] 59 12-223-207 C T [1-468] 60 12-225-541 C T[1-60], [368-598] 61 12-226-167 G C [1-255], [344-508] 62 12-226-458 C T[1-255], [344-508] 63 12-229-332 G C [1-456] 64 12-229-351 G C [1-456]65 12-230-364 C T [1-420] 66 12-231-100 C T [1-490] 67 12-231-148 C T[1-490] 68 12-231-266 C T [1-490] 72 10-239-368 C T [1-144], [373-618]73 10-223-30 G C [1-653], [729-1001] 74 10-223-72 A G [1-612],[688-1001] 75 10-223-130 A T [1-555], [631-1001] 76 10-223-262 A G[1-424], [500-1001] 77 10-223-392 A G [1-294], [370-1001] 78 10-224-341C T [137-176], [428-563], [920-1001] 82 10-251-128 A G [202-240],[373-415], [464-518], [581-777] 84 12-387-32 A G [1-396], [464-1001] 8510-242-316 G C [1-350], [418-1000] 86 10-245-412 A G [367-701] 8712-378-171 T C [1-731] 88 12-378-228 G A [1-788] 89 12-378-450 T A[1-1001] 90 12-379-65 A G [1-1001] 91 12-382-204 A G [1-1001] 9212-383-117 A G [1-37], [246-317], [383-1001] 93 12-383-170 A G[193-264], [330-1001] 94 12-383-268 G T [95-166], [232-1001] 9812-385-427 G T [257-826] 99 12-386-155 G T [272-682], [823-943] 10012-386-24 C T [272-682] 101 12-387-177 C T [1-251], [319-1001] 10212-389-431 C T [1-386], [470-583], [644-996] 103 12-391-366 C T[293-1001] 104 12-394-85 A C [1-103], [184-266], [345-1001] 10512-395-382 A G [1-885] 108 12-401-378 A G [1-880] 109 12-402-126 C T[99-823] 110 12-404-265 A G [1-261], [314-501], [715-733], [782-817] 11112-406-52 C T [136-952], [984-1001] 112 12-406-409 A G [1-595],[627-1001] 113 12-407-217 G C [247-673] 114 12-407-399 A T [1-491],[955-1001] 115 12-408-355 G C [80-907] 116 12-409-221 A C [1-500] 11712-410-301 C T [111-986] 118 10-395-101 A G [1-529], [633-1001] 11910-395-124 A G [1-539], [611-1001] 120 10-395-155 A T [1-509],[581-1001] 121 10-395-294 C T [1-371], [443-858] 122 10-396-100 A G[1-506], [635-776], [952-1001] 124 10-399-178 A G [1-142], [178-514],[632-1001] 125 10-400-369 A T [1-285], [385-513], [555-844], [878-941]126 10-392-20 A G [75-203], [245-534], [568-631], [746-849], [898-997]127 10-392-103 A G [1-552], [663-770], [819-1001] 128 10-392-324 G C[1-331], [442-549], [598-891], [977-1001] 129 10-393-27 G C [1-76],[187-294], [343-636], [722-1001] 130 10-393-324 A G [1-340], [423-1001]131 12-727-237 A G [513-1001] 132 12-728-224 A G [352-507], [661-772],[862-1001] 133 12-730-142 A G [1-1001] 134 12-730-193 A G [1-1001] 13512-731-60 C T [97-665], [711-729], [898-1001] 136 12-731-119 C T[1-606], [652-670], [839-1001] 137 12-731-137 G T [1-588], [634-652],[821-1001] 138 12-731-146 A C [1-579], [625-643], [812-1001] 13912-731-398 C T [1-327], [373-391], [560-743], [823-1001] 140 12-732-113A G [58-1001] 141 12-732-164 A G [1-1001] 142 12-732-165 G C [1-1001]143 12-732-445 C T [1-935], [975-1001] 144 12-734-201 T C [161-1001] 14512-735-42 G A [1-343], [374-566], [656-682], [731-961] 146 12-736-363 GA [1-1001] 147 12-737-69 T C [1-739] 148 12-737-296 G A [1-960] 14912-738-429 G A [1-205], [411-1001] 150 12-740-112 A G [1-26], [144-616],[743-1001] 151 12-740-118 C T [1-20], [138-610], [737-1001] 15212-741-265 G A [1-1001] 153 12-741-327 T A [1-1001] 154 12-741-376 G A[1-1001] 155 12-745-30 G A [1-1001] 156 12-745-75 T C [1-1001] 15712-745-343 T G [1-1001] 158 12-745-350 C A [1-1001] 159 12-746-320 C T[1-1001] 160 12-747-181 C T [1-1001] 161 12-747-302 C T [1-1001] 16412-752-37 G A [1-1003] 165 12-752-85 C G [1-1001] 166 12-752-196 T C[1-62], [108-1001] 167 12-752-484 T C [396-1001] 168 12-753-139 C T[1-1001] 169 12-753-376 C T [1-778], [855-1001] 170 12-754-172 C T[1-1001] 171 12-754-218 C T [1-1001] 172 12-754-328 G C [1-1001] 17312-754-396 G T [1-1001] 174 12-755-280 G C [1-1001] 176 12-758-257 A C[1-1001] 177 12-758-374 A C [1-1001] 178 12-758-424 A G [1-1001] 17912-761-23 G A [1-177], [253-701] 180 12-761-178 G A [1-292], [368-1001]181 12-764-329 G A [1-1001] 182 12-764-377 G A [1-1001] 183 12-765-168 GA [1-906] 184 12-765-504 T C [1-1002] 190 12-513-389 C T [1-1001] 19112-513-494 G C [1-999] 192 12-515-394 A T [77-950] 193 12-516-97 C T[1-744], [798-1001] 194 12-520-287 A T [179-468], [506-885] 19512-520-323 A G [143-432], [470-849] 196 12-523-179 G A [1-291],[344-1001] 197 12-523-270 G A [1-382], [435-1001] 198 12-527-367 T A[1-496], [595-1001] 199 12-529-376 T C [279-1001] 200 12-529-489 T C[1-37], [391-1001] 201 12-530-134 A T [1-94], [166-224], [316-803] 20212-530-393 C T [57-544], [766-1001] 203 12-531-173 C T [1-231],[414-735], [789-1001] 204 12-539-441 C T [1-1001] 205 12-543-78 G A[1-836] 206 12-543-79 C G [1-837] 207 12-546-235 C T [1-403], [492-1001]208 12-549-287 T C [149-494] 209 12-550-287 A G [304-1001] 21012-552-175 G A [1-750], [831-883] 211 12-554-330 G T [1-1001] 21212-556-312 A C [1-1001] 213 12-556-443 C T [1-1001] 214 12-558-205 C G[1-1001] 215 12-558-238 T C [1-1001] 216 12-558-305 T A [1-1001] 21712-769-39 G T [1-292], [593-624], [690-1001] 218 12-769-430 C T[202-233], [299-633] 219 12-770-73 G A [1-716] 220 12-772-200 G A[1-732], [788-1001] 221 12-772-254 T C [1-786], [842-1001] 23310-166-362 A C 250 12-86-79 G A [70-653], [748-1001] 252 12-89-369 G C[1-51], [102-1001] 253 12-89-91 A G [1-329], [380-1000] 254 12-94-210 CT [573-588] 255 12-94-516 A T [287-302] 256 12-96-64 C A [1-630],[936-1001] 257 12-97-83 A C [1-20], [543-649], [719-916], [964-1001] 25812-99-296 G A [1-210], [305-522], [904-1001] 259 12-100-266 T C[504-545], [927-949] 260 12-811-174 T C [1-945] 261 12-815-94 A G[1-1001] 262 12-815-383 A G [1-1001] 263 12-815-384 G C [1-1001] 26412-815-391 C T [1-1001] 268 12-821-62 T G [1-294], [376-437], [621-887]269 12-821-483 T G [1-48], [460-510], [664-715], [797-858] 27012-825-173 A C [1-34], [522-1001] 273 12-833-264 T A [1-86], [216-446],[558-1001] 274 12-833-279 G A [1-101], [231-461], [573-1001] 27512-833-280 T C [1-102], [232-462], [574-1001] 276 12-833-373 G A[1-195], [325-555], [667-1001] 277 12-834-183 A G [295-990] 27812-835-54 A G [1-1001] 279 12-836-134 C T [84-249], [354-587],[633-1001] 280 12-836-237 A G [1-147], [252-945] 281 12-836-238 A T[1-123], [228-919] 282 12-836-257 A G [1-123], [228-919] 283 12-836-275A C [1-108], [213-904] 284 12-838-179 A G [1-519], [718-1001] 28512-839-397 G A [1-43], [110-1001] 286 12-840-47 C G [1-553], [659-1001]287 12-840-77 T C [1-583], [689-1001] 288 12-841-445 G C [1-502] 29112-844-167 T C [186-1001] 292 12-845-364 G A [1-849] 293 12-846-209 A T[1-817] 294 12-847-123 A G [1-1001] 295 12-849-242 C A [1-27], [490-658]298 10-336-232 A G [507-1001] 299 12-102-104 A G [1-630], [712-790] 30012-102-111 A G [1-630], [712-790] 301 12-102-275 A G [1-581], [663-741],[834-851], [891-1001] 302 12-103-202 C T [188-767] 303 12-103-214 A G[176-755] 304 12-104-351 T G [1-20], [336-402], [438-511], [911-935] 30512-105-435 A G [1-147], [492-924] 306 12-109-149 A G [1-59], [289-607]307 12-109-197 A G [1-59], [289-607] 308 12-109-209 A G [1-59],[289-607] 309 12-109-284 A G [1-59], [289-607] 310 12-113-276 T C[1-1001] 311 12-115-57 A G [507-1001] 312 12-119-26 T C [1-569] 31412-354-334 G A [1-750] 315 12-357-140 C T [1-1001] 316 12-361-320 G T[1-201], [268-1001] 317 12-361-388 A G [1-133], [200-1001] 31812-365-251 G C [1-41], [132-151], [232-622], [688-933] 319 12-374-261 GA [249-1001] 321 10-311-274 C T [125-305], [472-878] 322 10-314-76 C T[1-224], [290-535], [803-1001] 335 10-30-349 A G 340 12-884-203 C T[1-349], [464-1001] 342 10-479-350 C T [1-280], [446-1001] 34310-479-394 A G [1-236], [402-1001] 345 12-854-64 A G [1-1001] 34612-854-472 G T [1-1001] 347 12-855-194 T G [1-1001] 348 12-855-288 T C[1-1001] 349 12-855-423 T G [1-1001] 350 12-857-25 C T [221-985] 35112-858-346 T C [1-1001] 352 12-858-443 G A [1-1001] 353 12-860-388 G A[1-30], [157-628], [831-1001] 354 12-861-270 C T [1-780] 355 12-862-349A G [78-1001] 356 12-862-365 C T [62-1001] 357 12-862-452 G T [1-1000]358 12-866-423 C T [1-434], [521-1001] 359 12-867-47 C T [81-769] 36012-868-181 A G [306-1001] 361 12-868-198 A G [289-1001] 362 12-868-282 CT [205-1001] 363 12-869-128 A C [1-128], [908-1001] 365 12-872-52 A G[436-1001] 366 12-872-293 A G [185-1001] 367 12-873-185 T C [114-257],[288-377], [572-1001] 368 12-873-319 T A [1-139], [248-391], [422-511],[706-1001] 369 12-875-248 T C [1-408], [525-1001] 370 12-876-265 T A[1-1001] 371 12-876-280 C G [1-1001] 372 12-876-454 G A [1-1001] 37312-877-59 C T [329-1001] 374 12-877-69 G T [319-1001] 375 12-877-79 C T[309-1001] 376 12-878-153 C T [207-937] 377 12-878-419 G T [1-629],[734-929] 378 12-879-67 G C [1-200], [261-460], [527-1001] 37912-879-439 A G [1-89], [156-796] 380 12-881-210 A G [1-1001] 38112-881-389 G T [1-841] 382 12-883-273 G C [1-56], [96-1001] 38312-885-196 T C [1-1001] 384 12-885-333 C G [1-1001] 385 12-885-407 T C[1-1001] 386 12-885-410 C G [1-1001] 387 12-886-195 T C [1-815],[867-1001] 388 12-886-348 T C [1-968] 389 12-887-201 G A [1-59],[181-1001] 390 12-887-467 T C [295-325], [447-1001] 391 12-888-98 G A[1-717], [916-1001] 392 12-888-203 C A [1-822] 393 12-888-315 T G[1-1001] 394 12-889-518 G A [1-89], [280-320], [441-1001] 395 12-894-266T C [1-1001] 396 12-895-391 G A [148-1001] 397 12-896-140 T A [60-76],[126-1001] 398 12-897-115 T C [259-557] 399 12-897-225 G A [369-667] 40012-898-49 G A [1-283], [372-781] 401 12-164-119 T G [1-646], [979-1001]403 12-168-365 C G [1-600] 407 12-175-214 A G [1-154], [227-317],[391-660], [747-1001] 408 12-177-183 C G [1-837], [975-1001] 40912-177-366 C A [1-1001] 427 10-47-103 A C 428 10-47-125 A T 43310-40-222 A G 434 10-40-252 C T 442 10-388-379 C T [1-202], [383-1001]443 10-389-116 A G [1-538], [693-1001] 444 10-389-349 C T [1-305],[460-1001] 445 10-391-94 A G [1-259], [301-575], [691-928] 44612-277-147 A T [1-693] 447 12-278-413 A G [1-151], [365-733], [775-1001]448 12-288-190 G A [1-701] 449 12-289-35 A G [1-791], [946-1001] 45012-296-119 A G [451-550] 451 12-297-291 C T [1-1001] 452 12-298-105 G A[1-162], [348-1001] 453 12-300-126 A G [1-782] 454 12-300-410 A C[1-415], [447-498] 455 12-301-379 A T [1-627], [932-1001] 456 12-302-264G A [1-1001] 458 12-310-105 G C [293-1001] 459 12-314-453 A T [1-392],[439-558], [643-799] 460 12-316-292 C T [1-460] 461 10-281-314 G T[1-282], [453-832], [921-1001] 462 10-268-381 C T [1-197], [383-895] 46312-54-297 C T [97-326], [404-518], [658-1001] 464 10-276-407 C T [1-97],[510-615], [954-1001] 465 12-44-50 T C [220-534], [918-1001] 46612-44-67 T C [237-551], [935-1001] 469 12-45-305 C T [1-63], [488-816]470 12-46-92 A G [83-1001] 471 12-47-132 C T [1-184], [457-685],[799-871], [987-1001] 472 12-47-61 C T [72-255], [528-756], [870-942]473 12-48-100 A G [1-1001] 474 12-48-323 A G [1-747] 475 12-48-369 C T[1-682] 476 12-48-37 C T [1-1001] 477 12-49-131 T C [1-609], [677-749],[920-1001] 478 12-49-53 G A [1-531 ], [599-671], [842-1001] 479 12-49-64G A [1-542], [610-682], [853-1001] 480 12-51-234 T C [1-47], [182-541],[919-1001] 481 12-51-253 C A [1-66], [201-560], [938-1001] 482 12-51-370G A [1-182], [317-676] 483 12-52-400 G A [1-100], [404-1001] 48412-57-192 G A [1-168], [286-752] 485 12-57-221 G A [1-197], [315-781]486 12-57-510 C A [1-163], [251-486], [604-1010] 494 10-13-152 C T 51012-561-270 C T [188-203], [496-642], [697-738] 511 12-563-87 C T [1-929]512 12-564-64 G T [1-213], [381-1001] 513 12-564-214 C T [1-64],[232-1001] 514 12-568-207 G T [424-513], [613-1001] 515 12-568-365 G T[266-355], [455-1001] 516 12-568-367 G T [264-353], [453-1001] 51712-571-337 G C [1-53], [327-897] 518 12-573-378 A G [1-335], [437-910]519 10-294-256 G C [1-53], [327-897] 520 10-294-304 G C [279-849],[942-1001] 522 10-296-80 A G [359-397], [531-906] 523 10-296-373 A G[60-105], [239-623], [924-1001] 524 10-298-122 C T [1-565], [737-873]525 10-298-158 A G [1-529], [701-837] 526 10-300-49 A G [285-643],[808-854] 527 10-300-185 C T [92-507], [672-718], [976-1001] 54912-63-402 A G [1-472] 550 12-63-74 A G [1-472] 551 12-64-271 C T [1-787]552 12-65-98 C T [112-272], [334-864] 553 12-70-147 A C [1-211],[491-1001] 554 12-70-397 C T [241-1001] 555 12-71-320 A G [1-1001] 55612-73-150 C T [1-140], [275-607], [646-821] 557 12-73-49 A G [1-240],[375-707], [746-921] 558 12-73-56 A T [1-233], [368-700], [739-914] 55912-74-38 G A [1-1001] 561 12-77-217 C T [1-822] 562 12-77-478 A G[1-562] 563 12-80-114 T C [1-1001] 564 12-80-233 G A [1-1001] 56512-82-250 A T [404-454] 571 12-214-85 CCTAT — [1-101], [259-305] 57212-215-272 T — [1-161], [254-499] 573 12-221-163 GTCCTCA T [1-64],[265-286] 574 12-225-82 T — [1-60], [368-598] 577 10-251-342 GG C[1-56], [156-301], [364-560] 578 10-395-367 A — [1-263], [367-717],[764-783] 579 12-730-58 ACAA — [162-251], [287-321 ], [517-767] 58012-735-208 — Deletion [1-689], [779-805], [854-1002] 581 12-739-22 G —[1-39], [386-640], [791-1002] 582 12-540-363 T — [1-1002] 583 12-550-206T — [380-1002] 587 12-834-290 G — [196-1002] 589 12-857-122 CTCT —[145-1002] 590 12-872-175 T — [1-41], [310-1102] 592 12-888-234 C —[1-850], [950-1002] 593 12-278-353 A — [1-208], [422-790], [832-1001]595 12-44-181 C — [308-622], [983-1002] 602 12-570-239 T C [386-671],[724-727], [947-1001] 603 12-570-344 T C [1-51], [491-601], [727-776],[829-832] 619 10-340-238 A G [231-310], [487-601] 620 10-342-301Insertion — [432-576], [605-609], [676-722] 621 10-342-373 C T[360-504], [533-537], [604-650], [930-1001] 625 10-346-23 A G [1-144],[233-274][305-347], [478-592], [696-945] 626 10-346-263 G C [1-37],[68-110], [241-355], [459-708] 627 10-346-305 C T [1-68], [199-313],[417-666], [961-1001] 629 10-350-332 C T [1-913] 630 10-350-72 C T[1-1001] 632 10-507-321 A C [1-308], [440-462], [552-652], [711-1000]633 10-507-353 C T [1-276], [408-430], [520-620], [679-1000] 63410-507-364 C T [1-265], [397-609], [668-1000] 635 10-507-405 C T[1-224], [356-378], [468-568], [627-1000] 636 10-508-191 C T [1-403],[442-444], [491-640], [942-1000] 637 10-508-245 C T [1-349], [388-390],[463-586], [888-1000] 638 10-510-173 ATTTA TTTTTT [243-380], [411-546]647 10-517-100 G C [1-1000] 648 10-518-125 G T [1-1000] 649 10-518-194 AG [1-1000] 650 10-522-71 A G [1-806], [844-863], [911-920], [950-1000]

[0758] TABLE 9 SEQ ID BIALLELIC ORIGINAL ALTERNATIVE NO. MARKER IDALLELE ALLELE 11 10-204-326 G A 12 10-32-357 C A 13 10-33-175 C T 1410-33-211 C T 15 10-33-234 A C 16 10-33-270 G A 17 10-33-327 T C 1810-34-290 G T 20 10-35-390 C T 21 10-36-164 G A 26 12-206-366 T C 2910-347-111 G C 30 10-347-165 C T 33 10-347-271 A T 34 10-347-348 G A 3610-349-47 T C 38 10-349-142 C G 41 10-339-32 C T 43 10-341-319 C T 4612-198-128 G A 47 12-206-81 G A 49 12-214-129 C T 50 12-214-151 G C 5112-214-360 G C 69 10-231-23 G A 70 10-233-386 A G 79 10-227-282 A G 8010-240-241 A G 83 10-252-209 G A 95 12-384-336 C T 96 12-384-451 G C 9712-385-123 C T 106 12-400-217 A G 107 12-400-280 A G 162 12-749-240 G A163 12-749-255 G T 175 12-757-384 T C 185 10-372-279 T C 186 10-375-136T C 187 10-376-281 A T 188 10-369-392 C T 222 10-87-73 C T 223 10-87-74A T 224 10-87-80 A G 225 10-87-140 C T 226 10-88-81 T C 227 10-89-41 G A228 10-90-35 G A 229 10-91-274 T G 231 10-94-197 G A 232 10-94-198 T G234 10-207-386 C G 235 10-207-409 G C 236 10-118-307 G A 237 10-173-247G A 238 10-173-294 A G 239 10-173-347 C T 240 10-103-104 C T 24110-103-323 T C 242 10-103-402 C T 243 10-106-98 C A 246 10-168-160 T C247 10-168-206 C A 248 10-168-284 T A 249 10-169-318 C A 251 12-88-393 AC 265 12-817-214 G A 266 12-817-355 T C 267 12-819-437 A G 27112-826-312 G A 272 12-831-59 G C 289 12-842-215 T C 290 12-842-447 A G297 10-336-137 T A 313 12-347-308 G A 320 10-308-116 C T 326 10-65-276 GA 327 10-67-42 A T 328 10-67-340 T C 331 10-59-176 C T 332 10-60-114 A G334 10-28-242 G A 336 10-181-42 C T 337 10-181-372 C T 338 10-183-260 CG 341 10-479-266 G A 364 12-870-491 A G 402 12-168-84 A C 404 12-170-299G A 405 12-171-360 C T 406 12-173-59 A G 410 10-128-45 T C 411 10-128-63A G 412 10-123-177 G A 414 10-120-137 G A 415 10-120-141 C A 42510-46-372 C T 429 10-48-184 C T 430 10-48-381 C T 431 10-49-33 C T 43210-39-148 A G 435 10-42-354 T C 436 10-154-42 C T 437 10-154-156 C T 43810-154-226 G A 439 12-776-259 A G 440 10-384-109 C T 441 12-296-388 A G457 12-309-405 A G 467 12-45-145 A G 468 12-45-166 G A 487 10-1-139 G T488 10-1-212 G T 489 10-1-241 C A 491 10-9-185 T C 492 10-9-264 C G 49310-11-22 T C 495 10-13-256 C T 496 10-13-282 T C 497 10-15-281 T G 49810-17-142 C T 499 10-18-302 C T 500 10-23-331 G A 501 10-25-152 T C 50210-25-258 C T 503 10-3-103 C T 504 10-3-144 T C 505 10-3-275 G T 50610-5-227 A C 507 10-7-155 T C 508 10-7-383 C T 509 10-7-98 G C 53310-20-274 A G 534 10-24-90 A C 536 10-24-221 G T 546 10-8-92 T C 54710-8-94 C T 548 12-61-472 C T 560 12-76-238 G T 566 10-176-85 C T 56710-176-51 C T 568 10-176-207 G T 569 10-176-397 C A 570 10-177-219 A C575 10-234-179 AA — 576 10-235-272 T — 584 10-207-410 — C 585 10-171-254GG — 586 12-94-110 — AATT 588 10-55-115 TTATA — 591 12-882-40 A — 59412-283-386 T — 598 10-485-256 A G 599 10-485-257 T C 600 10-474-320Insertion A — 601 10-387-371 T C 604 12-570-393 C T 605 12-570-421 T G606 12-570-62 Insertion TG — 607 10-4-144 C A 608 10-4-161 A C 60910-4-270 G C 610 10-4-340 A G 611 10-4-369 C T 612 10-4-420 G T 61310-13-396 Insertion AAT — 614 10-509-284 C T 616 10-339-124 C T 61710-340-112 C A 618 10-340-130 T A 622 10-343-231 Insertion C — 62310-343-278 C T 624 10-346-141 G A 628 10-349-216 Insertion CTG — 63110-507-170 A G 639 10-511-337 Deletion — 640 10-512-36 G C 641 10-511-62C T 642 10-512-318 G A 643 10-513-250 G A 644 10-513-262 T C 64510-513-352 G A 646 10-513-365 G A

[0759] TABLE 10 BIALLELIC 1^(ST) 2^(ND) SEQ ID NO. MARKER ID ALLELEALLELE 22 10-498-192 A G 31 10-347-203 A G 32 10-347-220 A G 3710-349-97 A G 39 10-349-224 G T 42 10-341-116 A G 81 10-249-185 A G 12310-397-201 G T 189 10-371-257 A C 230 10-93-133 C T 244 10-106-288 C T245 10-106-378 C T 296 10-336-58 C T 323 10-306-265 A G 324 10-52-386 CT 325 10-62-240 G C 329 10-55-265 C T 330 10-57-278 C T 333 10-27-176 AG 339 10-475-163 A G 344 10-482-145 A G 413 10-123-402 A G 416 10-179-39C T 417 10-180-65 G C 418 10-179-257 G T 426 10-46-36 T A 521 10-295-201G T 528 10-10-328 G A 529 10-12-52 C T 530 10-14-46 C T 532 10-20-111 AC 535 10-24-204 A G 537 10-24-234 A G 538 10-24-288 A G 539 10-24-311 GC 541 10-8-39 A C 542 10-8-120 A G 543 10-8-154 G C 544 10-8-101 A T 54510-8-86 C T 596 10-370-132 C T 597 10-370-254 C T 615 10-509-295Insertion and Deletion

[0760] TABLE 11 Sequences that are useful for designing some of theprimers and probes of the invention SEQ ID NO. POSITION RANGE OF NOVELSEQUENCE 26 [569-588], [815-956] 29 [1-97], [203-450], [747-956] 30[1-43], [149-396], [693-956] 31 [111-358], [655-956] 32 [94-341],[638-956] 33 [44-291], [588-956] 34 [1-214], [511-844] 36 [734-843] 37[684-793] 38 [639-748] 39 [557-666] 41 [217-319], [721-781] 42 [1-96],[276-387], [881-956] 43 [72-184], [678-820] 46 [1-56], [193-400] 47[855-874] 49 [1-101], [259-305] 50 [1-101], [259-305] 51 [1-101],[259-305] 79 [1-311], [512-1001] 80 [709-1001] 81 [1-231], [723-741] 83[291-476] 95 [1-138], [532-662], [970-1001] 96 [59-254], [648-778],[918-1001] 97 [318-757] 106 [88-182], [309-461], [798-843] 107 [1-119],[246-398], [735-780] 123 [1-449], [568-1001] 162 [264-407], [801-833]163 [249-392], [786-818] 175 [1-419] 185 [267-360], [549-599],[651-807], [851-1001] 186 [1-459], [691-1001] 187 [1-311], [557-1001]188 [1-155], [662-1001] 189 [1-39], [554-1001] 251 [746-1001] 265[315-445], [873-1001] 266 [174-304], [732-1001] 271 [1-173], [572-844],[884-917] 272 [1-75], [556-576] 289 [1-191] 290 [160-421] 296 [1-151],[681-1001] 297 [1-72], [602-1001] 313 [1-319] 320 [592-1001] 339 [1-24],[804-1001] 341 [1-364], [530-1001] 364 [1-270], [554-1001] 402 [1-319]404 [1-319], [767-830] 405 [1-222], [639-1001] 439 [1-73], [608-900] 440[1-40], [732-1001] 441 [182-281] 457 [1-315], [838-1001] 467 [1-222],[647-1001] 468 [1-201], [626-954] 521 [1-138], [281-412], [529-880] 548[60-80] 560 [539-810] 588 [406-418] 591 [150-320], [777-824], [864-1002]594 [300-450] 596 [196-237], [920-1001] 597 [74-115], [798-1001] 598[557-1001] 599 [556-1001] 600 [256-267], [669-670], [833-835] 604[1-100], [540-650], [776-825], [878-881], [969-985] 605 [1-128],[568-678], [804-853], [906-909], [997-1001] 606 [210-320], [446-495],[548-551], [771-1001] 607 [1-54] 608 [1-54] 609 [1-54] 610 [1-54] 611[1-54] 612 [1-54] 613 [1-30], [138-179] 614 [725-814] 615 [714-803] 616[1-252], [634-713], [890-1001] 617 [106-155], [357-436], [613-727] 618[88-137], [339-418], [595-709] 622 [138-178], [592-638], [863-1001] 623[91-131], [545-591], [816-1001] 624 [1-29], [118-159], [190-232],[363-477], [581-830] 628 [587-698] 631 [199-459], [591-613], [703-803],[862-1000] 641 [1-159], [190-325] 646 [1-20]

[0761] TABLE 12 Microsequencing primers POSITION RANGE OF COMPLEMENTARYPOSITION SEQ ID MICROSEQUENCING RANGE OF NO. PRIMERS MICROSEQUENCINGPRIMERS 1 458-477 479-498 2 459-477* 479-498 3 458-477 479-498 4 458-477479-498 5 458-477 479-498 6 458-477 479-498 7 458-477 479-498 8 458-477479-498 9 458-477 479-498 10 458-477 479-498 11 458-477 479-497* 12459-477* 479-498 13 459-477* 479-498 14 458-477 479-498 15 459-477*479-498 16 459-477* 479-498 17 459-477* 479-498 18 458-477 479-498 19459-477* 479-498 20 459-477* 479-498 21 458-477 479-497* 22 458-477479-498 23 458-477 479-497* 24 458-477 479-498 25 459-477* 479-498 26459-477* 479-498 27 458-477 479-498 28 458-477 479-498 29 458-477479-498 30 458-477 479-498 31 459-477* 479-498 32 458-477 479-497* 33458-477 479-498 34 458-477 479-498 35 458-477 479-498 36 458-477 479-49837 458-477 479-497* 38 458-477 479-498 39 458-477 479-497* 40 458-477479-498 41 458-477 479-498 42 458-477 479-497* 43 458-477 479-498 44100-118* 120-139 45 224-242* 244-263 46 108-127 129-148 47 458-477479-498 48  16-34*  36-55 49 110-128* 130-149 50 131-150 152-171 51338-357 359-378 52 446-465 467-486 53 398-417 419-437* 54 209-228230-248* 55 235-254 256-275 56 458-477 479-498 57 282-301 303-322 58159-178 180-199 59 188-206* 208-227 60 521-539* 541-560 61 147-165*167-185* 62 435-454 456-475 63 312-331 333-352 64 331-350 352-371 65344-363 365-384 66  79-98 100-119 67 127-146 148-167 68 245-264 266-28569 480-499 501-519* 70 481-500 502-520* 72 481-500 502-521 73 482-500*502-521 74 481-500 502-521 75 481-500 502-521 76 481-500 502-521 77481-500 502-521 78 481-500 502-521 79 481-500 502-520* 80 481-500502-521 81 481-500 502-521 82 481-500 502-521 83 481-500 502-521 84482-500* 502-521 85 480-499 501-520 86 481-500 502-521 87 481-500502-521 88 481-500 502-521 89 481-500 502-521 90 481-500 502-521 91482-500* 502-521 92 481-500 502-521 93 481-500 502-521 94 481-500502-521 95 481-500 502-521 96 481-500 502-521 97 238-257 259-278 98481-500 502-521 99 423-442 444-462* 100 293-312 314-333 101 481-500502-520* 102 481-500 502-521 103 481-500 502-521 104 481-500 502-521 105365-384 386-405 106 482-500* 502-521 107 481-500 502-521 108 360-379381-400 109 303-322 324-343 110 297-316 318-337 111 481-500 502-521 112481-500 502-521 113 481-500 502-521 114 481-500 502-521 115 482-500*502-521 116 209-228 230-249 117 466-485 487-506 118 481-500 502-521 119481-500 502-521 120 481-500 502-521 121 481-500 502-521 122 481-500502-521 123 481-500 502-521 124 481-500 502-521 125 481-500 502-521 126477-496 498-517 127 481-500 502-521 128 481-500 502-521 129 481-500502-521 130 481-500 502-521 131 481-500 502-521 132 481-500 502-521 133481-500 502-521 134 481-500 502-521 135 481-500 502-521 136 481-500502-521 137 481-500 502-521 138 481-500 502-521 139 481-500 502-521 140481-500 502-521 141 481-500 502-521 142 481-500 502-520* 143 481-500502-521 144 481-500 502-521 145 481-500 502-521 146 481-500 502-521 147482-500* 502-521 148 481-500 502-521 149 481-500 502-520* 150 482-500*502-521 151 481-500 502-521 152 481-500 502-521 153 481-500 502-521 154481-500 502-521 155 481-500 502-521 156 481-500 502-521 157 481-500502-521 158 481-500 502-521 159 481-500 502-521 160 481-500 502-521 161481-500 502-521 162 481-500 502-521 163 481-500 502-521 164 488-507509-528 165 481-500 502-521 166 481-500 502-521 167 481-500 502-521 168481-500 502-521 169 481-500 502-521 170 481-500 502-521 171 481-500502-521 172 481-500 502-521 173 481-500 502-521 174 481-500 502-521 175481-500 502-521 176 481-500 502-521 177 481-500 502-521 178 481-500502-521 179 521-540 542-561 180 481-500 502-521 181 481-500 502-521 182481-500 502-521 183 481-500 502-521 184 481-500 502-521 185 481-500502-521 186 481-500 502-521 187 481-500 502-521 188 481-500 502-521 189481-500 502-521 190 481-500 502-521 191 481-500 502-521 192 481-500502-521 193 481-500 502-520* 194 481-500 502-521 195 481-500 502-520*196 482-500* 502-521 197 481-500 502-521 198 481-500 502-520* 199481-500 502-521 200 481-500 502-521 201 482-500* 502-521 202 481-500502-521 203 481-500 502-520* 204 481-500 502-521 205 481-500 502-521 206481-500 502-521 207 481-500 502-521 208 481-500 502-521 209 481-500502-521 210 481-500 502-521 211 481-500 502-521 212 481-500 502-521 213481-500 502-521 214 481-500 502-521 215 481-500 502-521 216 481-500502-521 217 481-500 502-521 218 481-500 502-521 219 481-500 502-521 220481-500 502-521 221 481-500 502-521 222  52-71  73-92 223  53-72  74-93224  59-78  80-99 225 118-137 139-158 226  62-80*  82-101 227  21-40 42-61 228  15-34  36-54* 229 254-273 275-294 230 113-132 134-153 231178-196* 198-217 232 178-197 199-218 233 342-361 363-382 234 368-386*388-407 235 390-408* 410-429 236 287-306 308-326* 237 227-246 248-267238 274-293 295-313* 239 328-346* 348-367 240  84-103 105-124 241304-322* 324-343 242 383-402 404-423 243  78-97  99-118 244 269-287*289-308 245 361-379* 381-400 246 141-159* 161-180 247 187-205* 207-226248 263-282 284-303 249 297-316 318-337 250 481-500 502-520* 251 481-500502-521 252 482-500* 502-521 253 481-500 502-521 254 481-500 502-521 255501-520 522-541 256 482-500* 502-521 257 481-500 502-521 258 482-500*502-521 259 482-500* 502-521 260 481-500 502-521 261 481-500 502-521 262481-500 502-521 263 480-499 501-520 264 481-500 502-521 265 481-500502-521 266 481-500 502-521 267 481-500 502-521 268 481-500 502-521 269481-500 502-521 270 481-500 502-521 271 481-500 502-521 272 481-500502-521 273 481-500 502-521 274 481-500 502-521 275 482-501 503-522 276481-500 502-521 277 463-482 484-503 278 481-500 502-521 279 481-500502-521 280 480-499 501-520 281 456-475 477-496 282 478-497 499-518 283481-500 502-521 284 481-500 502-521 285 481-500 502-521 286 481-500502-521 287 481-500 502-521 288 425-444 446-465 289 481-500 502-521 290479-498 500-519 291 481-500 502-521 292 481-500 502-521 293 481-500502-521 294 481-500 502-521 295 481-500 502-521 296 481-500 502-521 297481-500 502-521 298 481-500 502-521 299 359-378 380-399 300 366-385387-406 301 481-500 502-521 302 482-500* 502-521 303 481-500 502-521 304481-500 502-520* 305 419-438 440-459 306 258-277 279-297* 307 306-325327-346 308 318-337 339-358 309 393-412 414-433 310 482-500* 502-521 311481-500 502-520* 312 482-500* 502-521 313 481-500 502-521 314 482-500*502-521 315 481-500 502-520* 316 481-500 502-520* 317 482-500* 502-521318 482-500* 502-521 319 482-500* 502-521 320 481-500 502-521 321481-500 502-521 322 481-500 502-521 323 481-500 502-521 324 366-385387-406 325 221-239* 241-260 326 256-275 277-295* 327  22-41  43-62 328322-340* 342-361 329 245-263* 265-284 330 259-277* 279-298 331 157-175*177-196 332  94-113 115-134 333 156-175 177-195* 334 222-241 243-261*335 330-349 351-369* 336  23-41*  43-62 337 355-373* 375-394 338 239-258260-279 339 481-500 502-521 340 482-500* 502-521 341 481-500 502-521 342481-500 502-521 343 481-500 502-521 344 481-500 502-521 345 481-500502-521 346 481-500 502-521 347 481-500 502-521 348 481-500 502-521 349481-500 502-521 350 456-475 477-496 351 482-500* 502-521 352 481-500502-521 353 481-500 502-521 354 481-500 502-521 355 481-500 502-521 356481-500 502-521 357 481-500 502-521 358 482-500* 502-521 359 481-500502-521 360 481-500 502-521 361 481-500 502-521 362 481-500 502-521 363481-500 502-521 364 481-500 502-521 365 481-500 502-521 366 481-500502-521 367 481-500 502-521 368 481-500 502-521 369 482-500* 502-521 370481-500 502-521 371 481-500 502-521 372 481-500 502-521 373 481-500502-521 374 481-500 502-521 375 481-500 502-521 376 481-500 502-521 377481-500 502-521 378 481-500 502-521 379 481-500 502-521 380 481-500502-521 381 481-500 502-521 382 481-500 502-521 383 481-500 502-521 384481-500 502-521 385 481-500 502-521 386 481-500 502-521 387 482-500*502-521 388 481-500 502-521 389 481-500 502-521 390 481-500 502-521 391481-500 502-521 392 482-500* 502-521 393 481-500 502-521 394 459-478480-499 395 481-500 502-521 396 481-500 502-520* 397 481-500 502-521 398481-500 502-521 399 481-500 502-521 400 508-527 529-548 401 481-500502-520* 402 482-500* 502-521 403 481-500 502-521 404 481-500 502-520*405 482-500* 502-521 406 481-500 502-520* 407 481-500 502-520* 408481-500 502-520* 409 481-500 502-521 410  26-44*  46-65 411  43-62 64-83 412 157-176 178-197 413 382-401 403-422 414 117-135* 137-156 415121-139* 141-160 416  19-38  40-59 417  46-64*  66-85 418 237-256258-276* 425 350-368* 370-389 426  15-34  36-55 427  82-101 103-122 428105-123* 125-144 429 164-182* 184-203 430 362-381 383-402 431  14-32* 34-53 432 130-149 151-169* 433 202-221 223-241* 434 230-249 251-270 435334-353 355-373* 436  22-41  43-62 437 137-155* 157-176 438 206-225227-246 439 481-500 502-521 440 481-500 502-521 441 482-500* 502-521 442481-500 502-521 443 481-500 502-521 444 481-500 502-521 445 481-500502-521 446 482-500* 502-521 447 482-500* 502-521 448 481-500 502-521449 481-500 502-521 450 481-500 502-521 451 481-500 502-521 452 481-500502-521 453 481-500 502-521 454 481-500 502-521 455 481-500 502-521 456481-500 502-521 457 481-500 502-521 458 481-500 502-521 459 482-500*502-521 460 481-500 502-520* 461 481-500 502-521 462 481-500 502-521 463482-500* 502-521 464 481-500 502-521 465 482-500* 502-521 466 481-500502-521 467 481-500 502-521 468 481-500 502-521 469 481-500 502-521 470481-500 502-520* 471 482-500* 502-521 472 481-500 502-521 473 481-500502-521 474 481-500 502-521 475 481-500 502-521 476 481-500 502-521 477482-500* 502-521 478 481-500 502-521 479 481-500 502-521 480 483-500*502-521 481 481-500 502-521 482 481-500 502-521 483 481-500 502-521 484481-500 502-520* 485 482-500* 502-520* 486 481-500 502-521 487 119-138140-158* 488 192-211 213-231* 489 222-240* 242-261 491 166-184* 186-205492 245-263* 265-284 493  2-21  23-42 494 133-151* 153-172 495 237-255*257-276 496 263-281* 283-302 497 261-280 282-301 498 123-141* 143-162499 282-301 303-322 500 311-330 332-351 501 133-151* 153-172 502 238-257259-278 503  84-102* 104-123 504 125-143* 145-164 505 255-274 276-294*506 208-226* 228-247 507 136-154* 156-175 508 361-380 382-401 509  78-97 99-118 510 481-500 502-520* 511 482-500* 502-521 512 481-500 502-520*513 481-500 502-521 514 481-500 502-521 515 481-500 502-521 516 481-500502-521 517 482-500* 502-521 518 482-500* 502-521 519 481-500 502-521520 481-500 502-521 521 481-500 502-521 522 481-500 502-521 523 481-500502-521 524 481-500 502-521 525 481-500 502-521 526 481-500 502-521 527481-500 502-521 528 307-326 328-346* 529  32-51  53-72 530  27-45* 47-66 532  91-109* 111-130 533 253-272 274-292* 534  71-89*  91-110 535184-203 205-223* 536 201-220 222-241 537 214-233 235-253* 538 268-287289-308 539 291-310 312-331 541  20-38*  40-59 542 100-119 121-140 543134-153 155-174 544  81-100 102-119* 545  67-85*  87-106 546  72-91 93-112 547  74-93  95-114 548 481-500 502-521 549 396-415 417-436 550 68-87  89-108 551 268-286* 288-307 552 419-438 440-459 553 482-500*502-521 554 482-500* 502-521 555 481-500 502-520* 556 481-500 502-521557 482-500* 502-521 558 481-500 502-521 559 481-500 502-520* 560481-500 502-520* 561 481-500 502-521 562 481-500 502-520* 563 481-500502-521 564 481-500 502-520* 565 230-249 251-270 566  66-84*  86-105 567 31-50  52-71 568 187-206 208-227 569 378-396* 398-416* 570 200-218*220-239 571  65-84 — 572 251-270 — 573 143-162 — 574  62-81 — 575195-213* — 576 471-490 — 577 478-497 — 578 477-496 — 579 478-497 — 580 —459-477* 581 — 498-516* 582 478-497 — 583 477-496 — 584 389-408 — 585235-254 — 586 479-497* — 587 478-497 — 588  95-113* — 589 478-497 — 590478-497 — 591 478-497 — 592 478-497 — 593 479-498 — 594 478-497 — 595438-457 — 596 481-500 502-521 597 481-500 502-521 598 481-500 502-521599 481-500 502-521 600 481-500 — 601 481-500 502-521 602 481-500502-521 603 481-500 502-521 604 481-500 502-521 605 481-500 502-521 606— 503-522 607 121-140 142-161 608 138-157 159-178 609 247-266 268-287610 317-336 338-357 611 346-365 367-386 612 397-416 418-437 613 374-395— 614 481-500 502-521 615 481-500 — 616 481-500 502-521 617 481-500502-521 618 481-500 502-521 619 481-500 502-521 620 481-500 — 621481-500 502-521 622 481-500 — 623 481-500 502-521 624 481-500 502-520*625 480-499 501-520 626 481-500 502-521 627 481-500 502-521 628 481-500— 629 481-500 502-521 630 481-500 502-521 631 481-500 502-521 632481-500 502-521 633 481-500 502-521 634 481-500 502-521 635 481-500502-521 636 481-500 502-521 637 481-500 502-521 638 481-500 — 639481-500 — 640 481-500 502-521 641 481-500 502-521 642 481-500 502-521643 481-500 502-521 644 481-500 502-521 645 481-500 502-521 646 481-500502-521 647 481-500 502-521 648 481-500 502-521 649 481-500 502-521 650481-500 502-521

[0762] TABLE 13 Amplification primers COMPLEMENTARY SEQ ID POSITIONRANGE OF POSITION RANGE OF NO. AMPLIFICATION PRIMERS AMPLIFICATIONPRIMERS 1 361-379 761-780 2 181-199 581-600 3 164-182 564-583 4 324-343536-553 5 294-310 743-760 6 221-237 670-687 7  70-86 519-536 8 326-343760-780 9 307-324 557-575 10 237-254 487-505 11 153-170 590-607 12121-139 522-541 13 304-322 705-723 14 268-286 669-687 15 245-263 646-66416 209-227 610-628 17 152-170 553-571 18 189-206 525-542 19 120-137526-543 20  88-105 494-511 21 315-334 741-760 22 287-306 621-638 23266-286 764-782 24 271-291 769-787 25 238-257 617-637 26 222-239 635-65427 140-157 553-572 28 405-422 826-845 29 368-385 789-808 30 314-331735-754 31 276-293 697-716 32 259-276 680-699 33 209-226 630-649 34132-149 553-572 35  90-109 488-507 36 432-451 829-848 37 382-401 779-79838 337-356 734-753 39 255-274 652-671 40 114-133 511-530 41 447-464845-864 42 363-380 771-789 43 160-177 568-586 44  1-20 450-469 45  1-19380-399 46  1-20 380-400 47 398-415 835-854 48  1-21 487-507 49  1-20429-448 50  1-20 429-448 51  1-20 429-448 52  1-20 479-499 53  1-20467-486 54  1-20 465-485 55  1-20 465-485 56  76-96 505-525 57  1-21387-407 58  1-20 449-468 59  1-20 449-468 60  1-19 581-598 61  1-19490-508 62  1-19 490-508 63  1-21 437-456 64  1-21 437-456 65  1-20401-420 66  1-19 470-490 67  1-19 470-490 68  1-19 470-490 69 478-495879-898 70 119-137 540-557 72 138-157 538-556 73 472-490 900-917 74431-449 859-876 75 374-392 802-819 76 243-261 671-688 77 113-131 541-55878 161-179 561-580 79 220-238 620-638 80 261-279 595-614 81 317-335720-738 82 374-393 732-751 83 293-312 701-720 84 470-488 901-921 85185-203 590-609 86  90-107 509-528 87  83-103 652-671 88 140-160 709-72889 361-381 930-949 90 437-456 885-905 91 298-318 727-747 92 385-404832-852 93 332-351 779-799 94 234-253 681-701 95 347-367 816-836 96463-483 932-952 97 136-155 591-611 98  75-94 530-550 99 290-310 669-688100 290-310 669-688 101 325-343 756-776 102  71-90 503-519 103 136-154571-590 104 417-435 930-949 105  5-23 530-549 106 285-304 749-769 107222-241 686-706 108  4-22 446-466 109 198-218 634-654 110  53-70 504-523111 450-468 881-899 112  93-111 524-542 113 285-303 746-766 114 103-121564-584 115 147-165 587-607 116  9-29 435-455 117 187-206 636-656 118406-425 797-816 119 384-403 775-794 120 354-373 745-764 121 216-235607-626 122 402-420 735-752 123 301-319 707-724 124 324-341 618-635 125134-153 533-552 126 478-495 906-924 127 399-416 827-845 128 178-195606-624 129 475-493 879-896 130 179-197 583-600 131 265-284 694-714 132277-295 720-740 133 360-378 867-887 134 309-327 816-836 135 442-462898-918 136 383-403 839-859 137 365-385 821-841 138 356-376 812-832 139104-124 560-580 140 389-408 879-898 141 338-357 828-847 142 337-356827-846 143  57-76 547-566 144 301-320 682-701 145  10-30 524-542 146386-406 844-862 147  47-67 547-566 148 268-288 768-787 149 478-498903-922 150 391-410 828-846 151 385-404 822-840 152 316-336 745-765 153378-398 807-827 154 427-447 856-876 155  67-86 512-530 156 112-131557-575 157 380-399 825-843 158 387-406 832-850 159 183-201 672-692 160321-340 767-787 161 200-219 646-666 162 262-281 761-780 163 247-266746-765 164  1-21 527-544 165  42-62 568-585 166 153-173 679-696 167441-461 967-984 168 364-382 900-920 169 127-145 663-683 170 330-349769-788 171 284-303 723-742 172 176-195 615-634 173 110-129 549-568 174222-242 688-708 175 118-135 628-647 176 245-264 749-768 177 128-147632-651 178  79-98 583-602 179  1-21 545-563 180 116-136 660-678 181355-373 811-828 182 403-421 859-876 183  74-94 649-668 184 408-428983-1002 185 224-242 627-646 186 366-385 765-784 187 221-239 646-665 188111-129 512-531 189 246-263 661-679 190 114-133 610-627 191  9-28505-522 192 107-126 537-557 193 405-424 891-911 194 216-234 653-673 195180-198 617-637 196 129-149 661-678 197 220-240 752-769 198 352-372849-867 199 349-369 856-875 200 461-481 968-987 201 369-389 798-817 202110-130 539-558 203 329-346 723-741 204  62-79 567-587 205 130-150558-578 206 131-151 559-579 207 267-284 795-815 208 304-324 770-787 209216-234 712-731 210 188-207 659-674 211 172-192 645-663 212 194-214674-693 213  63-83 543-562 214 236-256 687-705 215 268-288 719-737 216335-355 786-804 217 463-482 899-918 218  72-91 508-527 219 115-135555-573 220 236-256 681-700 221 290-310 735-754 222  1-18 345-362 223 1-18 345-362 224  1-18 345-362 225  1-18 345-362 226  1-18 349-368 227 1-19 401-420 228  1-18 409-427 229  1-18 402-420 230  1-19 407-426 231 1-19 403-420 232  1-19 403-420 233  1-18 363-380 234  1-12 444-464 235 1-12 444-464 236  1-18 343-361 237  1-19 418-435 238  1-19 418-435 239 1-19 418-435 240  1-19 420-439 241  1-19 420-439 242  1-19 420-439 243 1-19 406-424 244  1-19 406-424 245  1-19 406-424 246  1-18 354-371 247 1-18 354-371 248  1-18 354-371 249  1-20 408-425 250 130-149 562-579251 444-464 875-894 252 133-151 564-584 253 411-429 842-862 254 292-312819-839 255  6-26 533-553 256 101-121 547-564 257 419-437 866-886 258347-366 776-796 259 253-273 749-768 260 127-146 656-675 261 408-428849-859 262 119-139 560-570 263 118-138 559-569 264 111-131 552-562 265288-307 791-810 266 147-166 650-669 267  65-84 527-546 268  17-37542-562 269 438-458 963-983 270 329-347 774-794 271 337-357 792-811 272 92-112 540-559 273 315-335 746-764 274 330-350 761-779 275 331-351762-780 276 424-444 855-873 277 306-326 737-757 278 449-468 879-898 279368-386 909-929 280 266-284 807-827 281 242-260 783-803 282 242-260783-803 283 227-245 768-788 284 323-342 895-915 285 448-468 877-897 286 98-118 527-547 287 128-148 557-577 288  1-21 483-502 289 248-268697-715 290 478-498 927-945 291 245-265 649-667 292 415-435 843-863 293294-312 786-796 294 379-397 835-855 295 293-312 723-742 296 444-462845-863 297 365-383 766-784 298 270-288 671-689 299 276-296 733-753 300276-296 733-753 301 227-247 684-704 302 301-318 733-751 303 289-306721-739 304 373-393 831-851 305  5-23 442-462 306 130-148 577-597 307130-148 577-597 308 130-148 577-597 309 130-148 577-597 310 288-307756-775 311 445-463 901-921 312  41-61 509-526 313 195-213 636-656 314307-327 816-834 315 362-381 794-814 316 182-200 677-696 317 114-132609-628 318 252-270 697-717 319 262-280 741-761 320 386-403 754-771 321228-245 632-651 322 426-443 827-846 323 238-255 660-678 324  1-18412-429 325  1-20 419-438 326  1-19 408-425 327  2-20 403-422 328  2-20403-422 329  1-17 405-418 330  1-19 410-429 331  1-19 401-420 332  1-20409-428 333  1-18 290-307 334  3-20 410-429 335  1-18 368-385 336  1-18424-443 337  1-18 424-443 338  1-18 430-447 339 340-358 739-758 340299-317 817-837 341 236-253 638-657 342 152-169 554-573 343 108-125510-529 344 357-374 779-798 345 438-457 942-962 346  30-49 534-554 347234-254 674-694 348 328-348 768-788 349 463-483 903-923 350 452-471965-985 351 371-391 827-846 352 468-488 924-943 353 367-387 866-885 354232-251 668-688 355 153-171 607-626 356 137-155 591-610 357  50-68504-523 358  79-98 589-609 359 455-474 898-918 360 322-340 789-809 361305-323 772-792 362 221-239 688-708 363 374-394 813-833 364  11-31528-548 365 450-470 893-911 366 209-229 652-670 367 151-169 667-685 368285-303 801-819 369 251-271 727-747 370 242-261 745-765 371 257-276760-780 372 431-450 934-954 373 443-462 875-895 374 433-452 865-885 375423-442 855-875 376 349-369 839-859 377  83-103 573-593 378 435-453951-971 379  64-82 580-600 380 292-311 723-743 381 113-132 544-564 382229-248 674-694 383 235-255 677-696 384 372-392 814-833 385 446-466888-907 386 449-469 891-910 387 246-266 674-694 388 399-419 827-847 389184-204 683-701 390 450-470 949-967 391 148-168 578-598 392 253-273683-703 393 365-385 795-815 394 426-445 976-995 395 309-329 745-765 396348-368 870-889 397 138-158 622-640 398 106-126 595-615 399 216-236705-725 400  6-26 558-576 401 116-134 600-619 402 105-125 566-584 403386-406 847-865 404 296-315 781-799 405 142-160 521-541 406 443-460901-921 407 287-306 765-785 408 217-236 664-683 409 399-418 846-865 410 1-20 349-366 411  1-20 349-366 412  1-18 412-429 413  1-18 412-429 414 1-16 333-349 415  1-16 333-349 416  1-18 400-420 417  1-18 407-424 418 1-18 400-420 425  1-18 398-417 426  1-18 398-417 427  1-20 404-423 428 1-20 404-423 429  1-18 403-421 430  1-18 403-421 431  1-19 402-419 432 1-20 405-422 433  1-18 335-352 434  1-18 335-352 435  1-18 413-432 436 1-19 283-300 437  1-19 283-300 439 243-263 674-692 440 393-412 802-819441 114-134 543-563 442 123-141 542-561 443 386-403 792-809 444 153-170559-576 445 409-426 821-838 446 355-374 783-803 447  90-109 600-620 448199-219 670-690 449 467-486 915-935 450 383-403 812-832 451 211-229688-707 452  33-53 586-605 453 376-395 840-860 454  92-111 556-576 455124-142 553-573 456 286-306 745-764 457 456-475 884-904 458 397-415826-845 459  49-67 536-556 460 210-229 679-698 461 188-207 600-617 462121-138 521-540 463 210-228 647-667 464  95-113 511-530 465 147-166530-550 466 164-183 547-567 467 357-375 804-824 468 336-354 783-803 469198-216 645-665 470 410-429 792-811 471 370-388 756-776 472 441-459827-847 473 403-421 904-921 474 180-198 681-698 475 134-152 635-652 476466-484 967-984 477 180-200 613-630 478 102-122 535-552 479 113-133546-563 480 329-347 717-734 481 348-366 736-753 482 464-482 852-869 483451-471 881-900 484 151-168 672-692 485 180-197 701-721 486 469-486990-1010 487  1-20 429-448 488  1-20 429-448 489  1-20 429-448 491  1-18332-351 492  1-18 332-351 493  1-18 402-421 494  1-19 402-420 495  1-19402-420 496  1-19 402-420 497  1-20 409-428 498  1-20 413-431 499  1-19403-422 500  1-19 427-446 501  1-18 408-427 502  1-18 408-427 503  1-19411-430 504  1-19 411-430 505  1-19 411-430 506  1-20 404-421 507  1-20399-418 508  1-20 399-418 509  1-20 399-418 510 232-252 703-723 511415-433 862-882 512 438-456 920-940 513 289-307 771-791 514 295-313812-829 515 137-155 654-671 516 135-153 652-669 517 165-183 647-667 518124-144 590-610 519 246-264 650-669 520 198-216 602-621 521 301-320701-720 522 423-440 833-850 523 131-148 541-558 524 384-401 806-825 525348-365 770-789 526 453-470 802-820 527 317-334 666-684 528  1-20414-433 529  1-18 330-349 530  1-18 414-431 532  1-19 400-419 533  1-19400-419 534  1-20 416-435 535  1-20 416-435 536  1-20 416-435 537  1-20416-435 538  1-20 416-435 539  1-20 416-435 541  1-20 427-446 542  1-20427-446 543  1-20 427-446 544  1-20 427-446 545  1-20 427-446 546  1-20427-446 547  1-20 427-446 548  39-57 548-568 549  15-35 445-464 550 15-35 445-464 551  19-38 407-425 552 342-360 777-797 553 355-374784-804 554 105-124 534-554 555 187-206 703-721 556 353-370 799-818 557453-470 899-918 558 446-463 892-911 559  88-106 519-538 560 264-283671-689 561 288-306 772-792 562  28-46 512-532 563 101-121 595-614 564220-240 714-733 565  1-21 436-454 566  1-18 404-423 567  1-18 404-423568  1-18 404-423 569  1-18 404-423 570  1-18 400-419 571  1-20 429-448572  1-20 479-499 573  1-21 387-407 574  1-19 581-598 575  36-56 346-366576 220-237 553-571 577 157-176 515-534 578 140-159 531-550 579 441-459948-968 580 133-153 647-665 581 476-494 907-927 582 425-445 939-957 583292-310 788-807 584  1-18 443-463 585  1-19 333-351 586 389-409 916-936587 207-227 638-658 588  1-19 401-418 589 376-395 889-909 590 324-344767-785 591 459-478 904-924 592 281-301 711-731 593 147-166 657-677 594114-132 547-567 595 235-254 618-638 596 371-389 772-791 597 249-267650-669 598 246-264 652-669 599 245-263 651-668 600 182-199 590-609 601131-149 535-552 602 255-274 719-738 603 360-379 824-843 604 409-428873-892 605 437-456 901-920 606  79-98 543-562 607  1-18 427-444 608 1-18 427-444 609  1-18 427-444 610  1-18 427-444 611  1-18 427-444 613 1-19 402-420 614 218-235 618-637 615 207-224 607-626 616 379-396776-795 617 390-408 791-809 618 372-390 773-791 619 264-282 665-683 620201-220 606-623 621 129-148 534-551 622 271-288 684-703 623 224-241637-656 624 363-381 763-780 625 478-496 878-895 626 241-259 641-658 627199-217 599-616 628 286-305 683-702 629 172-189 513-532 630 430-447771-790 631 332-350 739-758 632 181-199 588-607 633 149-167 556-575 634138-156 545-564 635  97-115 504-523 636 311-328 727-746 637 257-274673-692 638 329-347 729-748 639 165-182 569-587 640 147-164 551-569 641440-457 844-862 642 184-203 607-626 643 252-269 660-677 644 240-257648-665 645 150-167 558-575 646 137-154 545-562 647 402-420 722-740 648378-396 630-648 649 309-327 561-579 650 431-449 885-903

[0763] TABLE 14 Preferred probes useful in genotyping eicosanoid-relatedbiallelic markers by hybridization assays. SEQ ID POSITION RANGE OF NO.PROBES 1 466-490 2 466-490 3 466-490 4 466-490 5 466-490 6 466-490 7466-490 8 466-490 9 466-490 10 466-490 11 466-490 12 466-490 13 466-49014 466-490 15 466-490 16 466-490 17 466-490 18 466-490 19 466-490 20466-490 21 466-490 22 466-490 23 466-490 24 466-490 25 466-490 26466-490 27 466-490 28 466-490 29 466-490 30 466-490 31 466-490 32466-490 33 466-490 34 466-490 35 466-490 36 466-490 37 466-490 38466-490 39 466-490 40 466-490 41 466-490 42 466-490 43 466-490 44107-131 45 231-255 46 116-140 47 466-490 48 23-47 49 117-141 50 139-16351 346-370 52 454-478 53 406-430 54 217-241 55 243-267 56 466-490 57290-314 58 167-191 59 195-219 60 528-552 61 154-178 62 443-467 63320-344 64 339-363 65 352-376 66  87-111 67 135-159 68 253-277 69488-512 70 489-513 72 489-513 73 489-513 74 489-513 75 489-513 76489-513 77 489-513 78 489-513 79 489-513 80 489-513 81 489-513 82489-513 83 489-513 84 489-513 85 488-512 86 489-513 87 489-513 88489-513 89 489-513 90 489-513 91 489-513 92 489-513 93 489-513 94489-513 95 489-513 96 489-513 97 246-270 98 489-513 99 431-455 100301-325 101 489-513 102 489-513 103 489-513 104 489-513 105 373-397 106489-513 107 489-513 108 368-392 109 311-335 110 305-329 111 489-513 112489-513 113 489-513 114 489-513 115 489-513 116 217-241 117 474-498 118489-513 119 489-513 120 489-513 121 489-513 122 489-513 123 489-513 124489-513 125 489-513 126 485-509 127 489-513 128 489-513 129 489-513 130489-513 131 489-513 132 489-513 133 489-513 134 489-513 135 489-513 136489-513 137 489-513 138 489-513 139 489-513 140 489-513 141 489-513 142489-513 143 489-513 144 489-513 145 489-513 146 489-513 147 489-513 148489-513 149 489-513 150 489-513 151 489-513 152 489-513 153 489-513 154489-513 155 489-513 156 489-513 157 489-513 158 489-513 159 489-513 160489-513 161 489-513 162 489-513 163 489-513 164 496-520 165 489-513 166489-513 167 489-513 168 489-513 169 489-513 170 489-513 171 489-513 172489-513 173 489-513 174 489-513 175 489-513 176 489-513 177 489-513 178489-513 179 529-553 180 489-513 181 489-513 182 489-513 183 489-513 184489-513 185 489-513 186 489-513 187 489-513 188 489-513 189 489-513 190489-513 191 489-513 192 489-513 193 489-513 194 489-513 195 489-513 196489-513 197 489-513 198 489-513 199 489-513 200 489-513 201 489-513 202489-513 203 489-513 204 489-513 205 489-513 206 489-513 207 489-513 208489-513 209 489-513 210 489-513 211 489-513 212 489-513 213 489-513 214489-513 215 489-513 216 489-513 217 489-513 218 489-513 219 489-513 220489-513 221 489-513 222 60-84 223 61-85 224 67-91 225 126-150 226 69-93227 29-53 228 23-47 229 262-286 230 121-145 231 185-209 232 186-210 233350-374 234 375-399 235 397-421 236 295-319 237 235-259 238 282-306 239335-359 240  92-116 241 311-335 242 391-415 243  86-110 244 276-300 245368-392 246 148-172 247 194-218 248 271-295 249 305-329 250 489-513 251489-513 252 489-513 253 489-513 254 489-513 255 509-533 256 489-513 257489-513 258 489-513 259 489-513 260 489-513 261 489-513 262 489-513 263488-512 264 489-513 265 489-513 266 489-513 267 489-513 268 489-513 269489-513 270 489-513 271 489-513 272 489-513 273 489-513 274 489-513 275490-514 276 489-513 277 471-495 278 489-513 279 489-513 280 488-512 281464-488 282 486-510 283 489-513 284 489-513 285 489-513 286 489-513 287489-513 288 433-457 289 489-513 290 487-511 291 489-513 292 489-513 293489-513 294 489-513 295 489-513 296 489-513 297 489-513 298 489-513 299367-391 300 374-398 301 489-513 302 489-513 303 489-513 304 489-513 305427-451 306 266-290 307 314-338 308 326-350 309 401-425 310 489-513 311489-513 312 489-513 313 489-513 314 489-513 315 489-513 316 489-513 317489-513 318 489-513 319 489-513 320 489-513 321 489-513 322 489-513 323489-513 324 374-398 325 228-252 326 264-288 327 30-54 328 329-353 329252-276 330 266-290 331 164-188 332 102-126 333 164-188 334 230-254 335338-362 336 30-54 337 362-386 338 247-271 339 489-513 340 489-513 341489-513 342 489-513 343 489-513 344 489-513 345 489-513 346 489-513 347489-513 348 489-513 349 489-513 350 464-488 351 489-513 352 489-513 353489-513 354 489-513 355 489-513 356 489-513 357 489-513 358 489-513 359489-513 360 489-513 361 489-513 362 489-513 363 489-513 364 489-513 365489-513 366 489-513 367 489-513 368 489-513 369 489-513 370 489-513 371489-513 372 489-513 373 489-513 374 489-513 375 489-513 376 489-513 377489-513 378 489-513 379 489-513 380 489-513 381 489-513 382 489-513 383489-513 384 489-513 385 489-513 386 489-513 387 489-513 388 489-513 389489-513 390 489-513 391 489-513 392 489-513 393 489-513 394 467-491 395489-513 396 489-513 397 489-513 398 489-513 399 489-513 400 516-540 401489-513 402 489-513 403 489-513 404 489-513 405 489-513 406 489-513 407489-513 408 489-513 409 489-513 410 33-57 411 51-75 412 165-189 413390-414 414 124-148 415 128-152 416 27-51 417 53-77 418 245-269 425357-381 426 23-47 427  90-114 428 112-136 429 171-195 430 370-394 43121-45 432 138-162 433 210-234 434 238-262 435 342-366 436 30-54 437144-168 438 214-238 439 489-513 440 489-513 441 489-513 442 489-513 443489-513 444 489-513 445 489-513 446 489-513 447 489-513 448 489-513 449489-513 450 489-513 451 489-513 452 489-513 453 489-513 454 489-513 455489-513 456 489-513 457 489-513 458 489-513 459 489-513 460 489-513 461489-513 462 489-513 463 489-513 464 489-513 465 489-513 466 489-513 467489-513 468 489-513 469 489-513 470 489-513 471 489-513 472 489-513 473489-513 474 489-513 475 489-513 476 489-513 477 489-513 478 489-513 479489-513 480 489-513 481 489-513 482 489-513 483 489-513 484 489-513 485489-513 486 489-513 487 127-151 488 200-224 489 229-253 491 173-197 492252-276 493 10-34 494 140-164 495 244-268 496 270-294 497 269-293 498130-154 499 290-314 500 319-343 501 140-164 502 246-270 503  91-115 504132-156 505 263-287 506 215-239 507 143-167 508 369-393 509  86-110 510489-513 511 489-513 512 489-513 513 489-513 514 489-513 515 489-513 516489-513 517 489-513 518 489-513 519 489-513 520 489-513 521 489-513 522489-513 523 489-513 524 489-513 525 489-513 526 489-513 527 489-513 528315-339 529 40-64 530 34-58 532  98-122 533 261-285 534  78-102 535192-216 536 209-233 537 222-246 538 276-300 539 299-323 541 27-51 542108-132 543 142-166 544  89-113 545 74-98 546  80-104 547  82-106 548489-513 549 404-428 550  76-100 551 275-299 552 427-451 553 489-513 554489-513 555 489-513 556 489-513 557 489-513 558 489-513 559 489-513 560489-513 561 489-513 562 489-513 563 489-513 564 489-513 565 238-262 56673-97 567 39-63 568 195-219 569 385-409 570 207-231 596 489-513 597489-513 598 489-513 599 489-513 601 489-513 602 489-513 603 489-513 604489-513 605 489-513 607 129-153 608 146-170 609 255-279 610 325-349 611354-378 612 405-429 614 489-513 616 489-513 617 489-513 618 489-513 619489-513 621 489-513 623 489-513 624 489-513 625 488-512 626 489-513 627489-513 629 489-513 630 489-513 631 489-513 632 489-513 633 489-513 634489-513 635 489-513 636 489-513 637 489-513 640 489-513 641 489-513 642489-513 643 489-513 644 489-513 645 489-513 646 489-513 647 489-513 648489-513 649 489-513 650 489-513

SEQUENCE LISTING INFORMATION

[0764] The Sequence Listing for this application is on duplicate compactdiscs labeled “Copy 1” and “Copy 2.” Copy 1 and Copy 2 each contain onlyone file named “Sequence-List.txt” which was created on Jun. 10, 2002,and is 1,243 KB. The entire contents of each of the computer discs areincorporated herein by reference in their entireties. TABLE 15 HAPLOTYPEFREQUENCY ANALYSIS MARKERS 10-253-298 10-33-175 10-33-234 10-33-32710-35-358 10-35-390 12-628-306 12-629-241 FLAP 5′ gene exon 2 intron 2intron 4 3′ gene cases/controls 287/186 295/174 295/274 295/270 291/280295/272 284/185 283/182 freq % case/controls 95/95 (C) 99/98 (C) 49/44(A) 78/76 (T) 72/69 (G) 31/23 (C) 88/90 (C) 76/72 (G) ESTIMATEDFREQUENCIES diff. freq. all. (cases - controls) 0.5 1.8 5.3 2.6 3.4 92,1 4,6 Frequencies haplotype p value 6.55E−01 1.35E−02 6.93E−022.94E−01 2.06E−01 2.29E−03 3.17E−01 1.14E−01 cases controls Odds ratioChi-S P value (1 df) 1 293 vs 265 A T 0.283 0.197 1.61 11.18 (8.2e−04) 2281 vs 177 A G 0.305 0.210 1.65 9.97 (1.6e−03) 3 293 vs 261 T T 0.3070.224 1.53 9.62 (1.8e−03) 4 289 vs 271 G T 0.304 0.231 1.46 7.77(5.2e−03) 5 293 vs 168 C T 0.309 0.226 1.53 7.26 (6.9e−03) 6 293 vs 265A T 0.276 0.208 1.46 7.17 (7.3e−03) 7 282 vs 178 T G 0.314 0.233 1.507.01 (7.7e−03) 37 281 vs 176 A T C 0.265 0.171 1.76 11.04 (8.6e−04) 38280 vs 173 A T G 0.292 0.194 1.71 10.71 (1.0e−03) 39 289 vs 264 A G T0.283 0.199 1.59 10.56 (1.1e−03) 40 278 vs 175 A C G 0.271 0.180 1.709.94 (1.6e−03) 41 284 vs 176 C A T 0.287 0.195 1.66 9.77 (1.7e−03) 121277 vs 171 A T C G 0.265 0.169 1.77 11.07 (8.6e−04) 122 278 vs 173 A G TG 0.290 0.195 1.69 10.29 (1.3e−03) 123 279 vs 176 A G T C 0.264 0.1751.70 9.80 (1.7e−03) 124 276 vs 175 A G C G 0.271 0.181 1.69 9.72(1.7e−03) 125 280 vs 174 C A T C 0.265 0.176 1.69 9.68 (1.8e−03) 247 275vs 171 A G T C G 0.265 0.170 1.77 10.91 (9.1e−04) 248 276 vs 169 C A T CG 0.265 0.172 1.74 10.30 (1.3e−03) 373 274 vs 169 C A G T C G 0.2650.172 1.73 10.13 (1.4e−03) 457 273 vs 163 C A T G T C G 0.247 0.167 1.647.74 (5.2e−03)

[0765] TABLE 16 HAPLOTYPE FREQUENCY ANALYSIS PERMUTATIONS TEST RESULTS(>1000 Iterations) Markers 10-33-234 10-35-390 intron 2 intron 4 ALT vsUS A T cases vs US controls 5.3 (51 vs 56) 6.93E−02 9 (31 vs 23)2.29E−03 ASSOCIATION diff all. p value diff all. p value Freq FreqHAPLOTYPE (AT) sample haplotype p- odds- chi-S P value PERMUTATIONSsizes frequencies excess ratio TEST RESULTS cases vs cases controls Av.Max >Iter/ controls Chi-S Chi-S nb of Iter. Asthmatics vs US 293 vs 2650.283 0.197 10.7 1.61 11.18 8.20E−04 1.2  7.4 0/1000  controls 1.2 12.91/10000

[0766] TABLE 17 HAPLOTYPE FREQUENCY ANALYSIS (Asthma) 297 Asthmatics vs186 US controls randoms MARKERS 12-208-35 12-226-167 12-206-36610-347-203 10-347-220 10-349-97 10-349-224 12-lipoxygenase 5′ geneintron 2 exon 6 exon 8 cases/controls 284/182 288/188 272/89 285/184274/184 282/182 271/177 frequency % (case/controls) 59/58 (T) 62/59 (C)57/62 (T) 57/58 (A) 58/60 (G) 59/60 (A) 57/60 (G) diff freq. all. (casescontrols) 0.9 3.4 −4.6 −1.2 −1.7 −1.9 −3.1 p value 7.52e−01 2.94e−012.73e−01 6.55e−01 5.84e−01 5.27e−01 3.43e−01 * * * * * * * 1 268 vs 176G 2 277 vs 174 A 3 274 vs 179 G 4 282 vs 176 A 5 280 vs 176 A 6 285 vs178 A 7 270 vs 176 C T 8 247 vs 86 C A T 9 255 vs 85 C G T 10 253 vs 84C G T 11 267 vs 172 A T 12 281 vs 181 C G 13 274 vs 182 C A 14 278 vs174 A 15 267 vs 175 G 16 276 vs 173 A 17 273 vs 172 A 18 268 vs 172 G 19261 vs 172 G 20 271 vs 171 A 21 277 vs 169 A 22 280 vs 171 A 23 264 vs170 G A 24 264 vs 81 T C G MARKERS 12-196-119 12-214-129 12-216-42112-219-230 12-223-207 12-lipoxygenase cases/controls 281/184 282/181288/182 288/187 287/186 frequency % 70/71 (T) 61/61 (T) 61/64 (G) 64/68(A) 62/62 (T) (case/controls) diff freq. all. −1.2 −0.7 −2.9 −4.2 0.8ESTIMATED FREQUENCIES (cases controls) haplotype p value 6.55e−017.52e−01 3.71e−01 1.80e−01 7.52e−01 frequencies Odds * * * * casescontrols p-excess ratio Chi-S P value (1 df) 1 268 vs 176 C G 0.1230.040 8.63 3.38 17.85 (2.3e−05) 2 277 vs 174 C G 0.125 0.041 8.71 3.3117.75 (2.5e−05) 3 274 vs 179 A G 0.123 0.041 8.49 3.26 17.47 (2.9e−05) 4282 vs 176 A G 0.125 0.043 8.57 3.20 17.29 (3.2e−05) 5 280 vs 176 C G0.115 0.037 8.08 3.36 16.81 (3.9e−05) 6 285 vs 178 A G 0.113 0.039 7.733.16 15.62 (7.4e−05) 7 270 vs 176 T 0.130 0.055 7.98 2.58 13.40(2.5e−04) 8 247 vs 86 0.405 0.256 19.96 1.97 12.10 (5.0e−04) 9 255 vs 850.406 0.259 19.81 1.95 11.80 (5.6e−04) 10 253 vs 84 0.399 0.253 19.621.97 11.73 (5.9e−04) 11 267 vs 172 T 0.088 0.030 5.97 3.09 11.45(7.0e−04) 12 281 vs 181 T 0.136 0.066 7.50 2.22 11.10 (8.6e−04) 13 274vs 182 T 0.137 0.067 7.52 2.21 11.05 (8.6e−04) 14 278 vs 174 T A G 0.1180.031 9.01 4.18 21.01 (4.4e−06) 15 267 vs 175 T A G 0.124 0.035 9.273.92 20.87 (4.8e−06) 16 276 vs 173 T A G 0.124 0.035 9.23 3.91 20.65(5.4e−06) 17 273 vs 172 T C G 0.121 0.034 9.01 3.90 20.02 (7.3e−06) 18268 vs 172 C A G 0.124 0.036 9.14 3.76 19.84 (8.2e−06) 19 261 vs 172 T CG 0.126 0.037 9.20 3.74 19.81 (8.2e−06) 20 271 vs 171 T C G 0.125 0.0379.11 3.69 19.49 (1.0e−05) 21 277 vs 169 C A G 0.125 0.038 9.06 3.6419.10 (1.2e−05) 22 280 vs 171 C A G 0.116 0.033 8.56 3.81 18.76(1.5e−05) 23 264 vs 170 C G 0.125 0.040 8.91 3.45 18.15 (2.0e−05) 24 264vs 81 G 0.197 0.056 14.96 4.13 18.01 (2.1e−05)

[0767] TABLE 18A ALLELE FREQUENCY ANALYSIS (Asthma) CASES (297 ALT) vsCONTROLS (186 US CAUCASIAN) MARKERS 12-197/244 12-208/35 12-226/16712-206/366 10-346/141 PROTEIN 12-LO 5′ gene In2 ex5 cases/controls277/180 284/182 288/188 272/89 285/185 frequency % (case/controls) 66/67(T) 58/57 (T) 62/58 (C) 57/61 (T) 99/100 (G) diff freq. all. (cases -controls) −1.0 0.9 3.4 −4.6 −0.4 p value 7.52e−01 7.52e−01 2.94e−012.73e−01 HOM * * * * Test Hardy cases vs 0.034 (HWD) −0.002 (HWE) −0.001(HWE) −0.014 (HWE) 0.000 (HWD) Weinberg controls 0.054 (HWD) −0.020(HWE)   0.022 (HWE)   0.000 (HWE) 0.000 (HWD) MARKERS 10-347/11110-347/165 10-347/203 10-347/220 10-349/97 10-349/224 PROTEIN 12-LO ex6ex8 cases/controls 284/180 268/185 280/184 283/184 287/182 277/177frequency % (case/controls) 99/100 (G) 99/100 (C) 57/58 (A) 57/59 (G)59/60 (A) 56/60 (G) diff freq. all. (cases - controls) −0.2 −0.2 −1.1−2.1 −1.4 −4.1 p value HOM 5.92e−01# 6.55e−01 4.80e−01 6.55e−012.06e−01 * * * * * Test Hardy cases vs 0.000 (HWD) 0.000 (HWD) −0.011(HWE) −0.005 (HWE) 0.003 (HWE) −0.010 (HWE) Weinberg controls 0.000(HWD) 0.000 (HWD)   0.012 (HWE)   0.021 (HWE) 0.008 (HWE) −0.004 (HWE)MARKERS 10-341/116 12-196/119 12-214/129 12-216/421 12-219/23012-223/207 PROTEIN 12-LO ex14 markers in bac cases/controls 286/176281/184 282/181 288/182 288/187 287/186 frequency % (case/controls)89/89 (G) 69/70 (T) 60/61 (T) 61/64 (G) 63/67 (A) 62/61 (T) diff freq.all. (cases - controls) 0.1 −1.2 −0.7 −2.9 −4.2 0.8 p value 7.52e−016.55e−01 7.52e−01 3.71e−01 1.80e−01 7.52e−01 * * * * * Test Hardy casesvs −0.008 (HWE) 0.012 (HWE) −0.013 (HWE) −0.012 (HWE) −0.010 (HWE)  0.012 (HWD) Weinberg controls −0.000 (HWE) 0.030 (HWE)   0.016 (HWE)  0.024 (HWE) −0.001 (HWE) −0.019 (HWD)

[0768] TABLE 18B HAPLOTYPE FREQUENCY ANALYSIS (Asthma) CASES (297 ALT)vs CONTROLS (186 US CAUCASIAN) ESTIMATED FREQUENCIES PERMUTATIONSHaplotype TEST RESULTS frequencies p- Odds Av. Max >Iter/ Marker 1Marker 2 Marker 3 Marker 4 Haplotype cases controls excess ratio Chi-S Pvalue (1 df) Chi-S Chi-s nb of Iter haplotype 1 PT2 265 vs 86 12-206/36610-349/224 CT 0.424 0.265 21.72 2.05 13.97 (1.8e−04) **** 2.1 8.3[0/100] haplotype 2 PT2 267 vs 89 12-206/366 10-347/220 CA 0.423 0.27420.56 1.94 12.55 (3.9e−04) *** 2.3 9.3 [0/100] haplotype 3 PT2 266 vs 8812-206/366 10-347/203 CG 0.421 0.277 19.96 1.90 11.64 (6.3e−04) *** 2.17.1 [0/100] haplotype 4 PT2 271 vs 87 12-206/366 10-349/97 CG 0.4080.270 18.88 1.86 10.69 (1.1e−03) *** 1.7 5.9 [0/100] haplotype 5 PT2 271vs 174 12-197/244 12-214/129 CC 0.148 0.077 7.66 2.08 10.03 (1.5e−03)*** 1.8 9.7 [0/100] haplotype 6 PT2 285 vs 175 10-341/116 12-223/207 AT0.042 0.008 3.45 5.48 8.95 (2.7e−03) *** 1.4 9.9 [2/100] haplotype 7 PT3282 vs 174 10-349/97 12-214/129 12-219/230 ACG 0.125 0.041 8.73 3.3217.87 (2.3e−05) ***** 1.8 8.5 [0/100] haplotype 8 PT3 287 vs 17610-349/97 12-216/421 12-219/230 AAG 0.126 0.043 8.67 3.23 17.65(2.6e−05) ***** 1.5 13.6 [0/100] haplotype 9 PT3 277 vs 176 10-347/22012-214/129 12-219/230 GCG 0.119 0.040 8.28 3.27 16.90 (3.7e−05) **** 3.015.4 [0/100] haplotype 10 PT3 275 vs 176 10-347/203 12-214/12912-219/230 ACG 0.115 0.037 8.05 3.35 16.64 (4.4e−05) **** 2.4 19.2[1/100] haplotype 11 PT3 283 vs 179 10-347/220 12-216/421 12-219/230 GAG0.119 0.041 8.11 3.15 16.40 (5.1e−05) **** 2.6 14.4 [0/100] haplotype 12PT3 266 vs 171 12-197/244 10-347/203 12-214/129 CAC 0.070 0.012 5.966.46 15.97 (6.3e−05) **** 1.9 11.3 [0/100] haplotype 13 PT3 248 vs 8512-206/366 10-347/165 10-349/224 CCT 0.427 0.255 23.11 2.18 15.86(6.7e−05) **** 2.7 18.8 [1/100] haplotype 14 PT3 271 vs 168 12-197/24410-349/97 12-214/129 CAC 0.069 0.012 5.83 6.37 15.34 (8.7e−05) **** 1.810.4 [0/100] haplotype 15 PT3 261 vs 86 12-206/366 10-347/220 10-349/224CAT 0.423 0.256 22.45 2.13 15.27 (9.2e−05) **** 2.4 7.8 [0/100]haplotype 16 PT3 276 vs 176 12-226/167 10-349/224 12-223/207 CTT 0.1370.055 8.64 2.72 15.27 (9.2e−05) **** 1.4 7.1 [0/100] haplotype 17 PT3280 vs 178 10-347/203 12-216/421 12-219/230 AAG 0.112 0.039 7.59 3.1215.15 (9.7e−05) **** 2.2 21.1 [2/100] haplotype 18 PT3 268 vs 17012-197/244 10-347/220 12-214/129 CGC 0.067 0.012 5.60 6.10 14.68(1.3e−04) **** 2.0 12.7 [0/100] haplotype 19 PT3 249 vs 88 12-206/36610-347/165 10-347/220 CCA 0.428 0.265 22.14 2.07 14.54 (1.3e−04) ****2.8 9.4 [0/100] haplotype 20 PT3 264 vs 86 12-206/366 10-346/14110-349/224 CGT 0.426 0.265 21.94 2.06 14.23 (1.6e−04) **** 2.6 11.2[0/100] haplotype 21 PT3 261 vs 85 12-206/366 10-347/203 10-349/224 CGT0.418 0.259 21.40 2.05 13.71 (2.0e−04) **** 2.2 8.3 [0/100] haplotype 22PT3 264 vs 84 12-206/366 10-349/97 10-349/224 CGT 0.411 0.253 21.19 2.0613.68 (2.1e−04) **** 2.4 7.2 [0/100] haplotype 23 PT3 248 vs 8712-206/366 10-347/165 10-347/203 CCG 0.425 0.268 21.54 2.03 13.55(2.3e−04) **** 2.2 8.5 [0/100] haplotype 24 PT3 261 vs 86 12-206/36610-347/111 10-349/224 CGT 0.421 0.265 21.30 2.02 13.43 (2.4e−04) ****2.1 6.1 [0/100] haplotype 25 PT3 268 vs 164 12-197/244 10-347/11112-214/129 CGC 0.151 0.068 8.89 2.44 13.33 (2.5e−04) **** 1.9 25.3[1/100] haplotype 26 PT3 265 vs 89 12-206/366 10-346/141 10-347/220 CGA0.426 0.274 21.00 1.97 13.04 (3.0e−04) **** 2.3 7.0 [0/100] haplotype 27PT4 280 vs 173 10-349/97 12-196/119 12-216/421 12-219/230 ATAG 0.1240.035 9.20 3.90 20.63 (5.4e−06) ***** 1.5 11.6 [0/100] haplotype 28 PT4274 vs 174 10-347/203 12-196/119 12-216/421 12-219/230 ATAG 0.117 0.0318.89 4.14 20.59 (5.7e−06) ***** 2.7 19.0 [0/100] haplotype 29 PT4 275 vs171 10-349/97 12-196/119 12-214/129 12-219/230 ATCG 0.126 0.037 9.213.72 19.86 (8.2e−06) ***** 2.0 11.8 [0/100] haplotype 30 PT4 276 vs 17510-347/220 12-196/119 12-216/421 12-219/230 GTAG 0.121 0.035 8.92 3.8019.84 (8.2e−06) ***** 2.4 14.7 [0/100] haplotype 31 PT4 269 vs 17210-347/203 12-196/119 12-214/129 12-219/230 ATCG 0.120 0.034 8.90 3.8619.61 (9.1e−06) ***** 2.4 12.2 [0/100] haplotype 32 PT4 280 vs 16510-349/97 10-341/116 12-214/129 12-219/230 AGCG 0.127 0.038 9.30 3.7319.55 (9.5e−06) ***** 1.9 16.0 [0/100] haplotype 33 PT4 270 vs 17210-347/220 12-196/119 12-214/129 12-219/230 GTCG 0.124 0.037 9.00 3.6819.31 (1.1e−05) ***** 2.8 19.3 [0/100] haplotype 34 PT4 282 VS 16910-349/97 12-214/129 12-216/421 12-219/230 ACAG 0.124 0.038 9.00 3.6218.98 (1.3e−05) ***** 1.8 10.5 [0/100] haplotype 35 PT4 267 vs 16712-197/244 12-208/35 12-214/129 12-223/207 CTCC 0.055 0.000 5.49 100.0018.96 (1.3e−05) ***** 2.6 21.7 [2/100] haplotype 36 PT4 285 vs 16710-349/97 10-341/116 12-216/421 12-219/230 AGAG 0.127 0.039 9.12 3.5518.89 (1.4e−05) ***** 2.0 12.3 [0/100] haplotype 37 PT4 277 vs 17210-347/220 12-214/129 12-216/421 12-219/230 GCAG 0.120 0.036 8.71 3.6218.59 (1.6e−05) ***** 2.3 25.8 [1/100] haplotype 38 PT4 275 vs 17110-347/203 12-214/129 12-216/421 12-219/230 ACAG 0.116 0.033 8.52 3.8018.57 (1.6e−05) ***** 2.7 21.6 [1/100] haplotype 39 PT4 276 vs 16212-208/35 10-341/116 12-214/129 12-219/230 AGCG 0.054 0.000 5.40 0.0018.10 (2.0e−05) ***** 2.7 18.6 [1/100] haplotype 40 PT4 245 vs 8512-206/366 10-347/165 10-347/220 10-349/224 CCAT 0.429 0.246 24.18 2.3017.77 (2.5e−05) ***** 3.0 7.5 [0/100] haplotype 41 PT4 268 vs 8112-208/35 12-206/366 10-349/97  12-216/421 TCGG 0.196 0.056 14.80 4.0917.76 (2.5e−05) ***** 1.8 15.1 [0/100]

[0769] TABLE 19 HAPLOTYPE FREQUENCY ANALYSIS (Zyflo secondary effects)89 ALT+ vs 208 ALT− MARKERS 12-208-35 12-226-167 12-206-366 10-347-20310-347-220 10-349-97 10-349-224 12-lipoxygenase 5′ gene intron 2 exon 6exon 8 Size 87/197 89/119 86/186 88/197 86/188 86/196 86/185(cases/controls) frequency % 58/59 (T) 61/63 (C) 55/58 (T) 56/58 (A)56/59 (G) 58/59 (A) 54/59 (G) (case/controls) diff freq. all. −0.8 −2.1−3.7 −2.2 −3.0 −1.4 −4.8 (cases - controls) p value 7.52e−01 5.84e−014.03e−01 5.84e−01 4.80e−01 7.52e−01 2.73e−01 * * * * * * * 1 87 vs 197 AG 2 83 vs 184 A 3 85 vs 185 T 4 85 vs 186 C 5 85 vs 179 T 6 85 vs 180 C7 86 vs 188 A 8 82 vs 174 C T 9 85 vs 179 T 10 83 vs 177 A T 11 82 vs183 G T 12 85 vs 183 G T 13 82 vs 168 C T 14 84 vs 175 C A 15 84 vs 184C G 16 85 vs 180 C 17 82 vs 181 T 18 83 vs 187 A 19 83 vs 171 A T 20 83vs 174 C T 21 82 vs 178 G T 22 82 vs 168 C T 23 82 vs 172 C G T 24 81 vs166 C A T 25 80 vs 171 C T MARKERS 12-196-119 12-214-129 12-216-42112-219230 12-223-207 12-lipoxygenase Size 86/195 89/193 89/199 89/19988/199 (cases/controls) frequency % 72/69 (T) 59/61 (T) 58/63 (G) 67/62(A) 62/63 (T) (case/controls) diff freq. all. 3.4 −2.4 −4.7 4.5 −0.6ESTIMATED FREQUENCIES (cases - controls) haplotype p value 4.03e−015.84e−01 2.73e−01 2.94e−01 7.52e−01 frequencies Odds P value * * * *cases controls ratio Chi-S (1 df) 1 87 vs 197 0.123 0.070 1.87 4.34(3.6e−02) 2 83 vs 184 C 0.209 0.140 1.63 4.09 (4.3e−02) 3 85 vs 185 A T0.151 0.048 3.53 16.76 (4.2e−05) 4 85 vs 186 A T 0.148 0.059 2.77 11.62(6.3e−04) 5 85 vs 179 C T 0.151 0.066 2.52 9.85 (1.7e−03) 6 85 vs 180 CT 0.149 0.070 2.34 8.42 (3.6e−03) 7 86 vs 188 A T 0.157 0.076 2.25 8.36(3.8e−03) 8 82 vs 174 A T 0.158 0.040 4.56 21.85 (2.9e−06) 9 85 vs 179 CA T 0.157 0.052 3.37 16.03 (6.0e−05) 10 83 vs 177 A T 0.162 0.057 3.2215.30 (9.2e−05) 11 82 vs 183 A T 0.147 0.050 3.23 14.18 (1.6e−04) 12 85vs 183 A T 0.143 0.049 3.21 13.97 (1.8e−04) 13 82 vs 168 C T 0.156 0.0583.02 13.08 (3.0e−04) 14 84 vs 175 A T 0.155 0.062 2.78 11.77 (5.9e−04)15 84 vs 184 A T 0.147 0.059 2.74 11.22 (7.8e−04) 16 85 vs 180 C A T0.145 0.058 2.74 11.05 (8.6e−04) 17 82 vs 181 T A T 0.132 0.050 2.8510.62 (1.1e−03) 18 83 vs 187 T T T 0.148 0.066 2.48 9.50 (2.1e−03) 19 83vs 171 C T 0.162 0.074 2.41 9.29 (2.3e−03) 20 83 vs 174 A A 0.111 0.0412.92 9.24 (2.3e−03) 21 82 vs 178 C T 0.152 0.068 2.45 9.22 (2.3e−03) 2282 vs 168 C A T 0.161 0.043 4.27 20.43 (6.0e−06) 23 82 vs 172 A T 0.1460.040 4.07 18.03 (2.1e−05) 24 81 vs 166 A T 0.160 0.047 3.82 17.77(2.5e−05) 25 80 vs 171 T A T 0.137 0.037 4.17 17.18 (3.4e−05)

[0770] TABLE 20A ALLELE FREQUENCY ANALYSIS (Zyflo secondary effects)CASES (85 ALT+) vs CONTROLS (208 ALT−) MARKERS 12-197/244 12-208/3512-226/167 12-206/366 10-346/141 PROTEIN 12-LO 5′gene in2 ex5 cases /controls 81/196 87/197 89/199 86/186 88/197 frequency % (case/controls)70/65(T) 58/59(T) 61/63(C) 55/58(T) 100/99(G) diff freq. all. (cases -controls) 5.8 −0.8 −2.1 −3.7 0.5 p value 1.80e−01 7.52e−01 5.84e−014.03e−01 HOM * * * * Test Hardy cases vs −0.001 (HWE)   0.008 (HWE)−0.020 (HWE) −0.031 (HWE) 0.000 (HWD) Weinberg controls   0.048 (HWD)−0.007 (HWE)   0.007 (HWE) −0.007 (HWE) 0.000 (HWD) MARKERS 10-347/11110-347/165 10-347/203 10-347/220 10-349/97 10-349/224 PROTEIN 12-LO ex6ex8 cases / controls 88/196 69/199 83/197 87/196 89/198 83/194 frequency% (case/controls) 99/100(G) 100/99(C) 56/58(A) 43/57(G) 59/59(A)54/57(G) diff freq. all. (cases - controls) −0.6 0.3 −1.8 0.1 −0.1 −3.0p value HOM 7.43e−01# 6.55e−01 7.52e−01 7.52e−01 4.80e−01 * * * * * TestHardy cases vs 0.000 (HWD) 0.000 (HWD) −0.037 (HWE) −0.020 (HWE) 0.000(HWE) −0.029 (HWE) Weinberg controls 0.000 (HWD) 0.000 (HWD)   0.000(HWE)   0.002 (HWE) 0.000 (HWE) −0.003 (HWE) MARKERS 10-341/11612-196/119 12-214/129 12-216/421 12-219/230 12-223/207 PROTEIN 12-LOex14 markers in bac cases / controls 89/197 86/195 89/193 89/199 89/19988/199 frequency % (case/controls) 90/89(G) 72/69(T) 59/61(T) 58/63(G)67/62(G) 62/63(T) diff freq. all. (cases - controls) 1.6 3.4 −2.4 −4.74.5 −0.6 p value 5.27e−01 4.03e−01 5.84e−01 2.73e−01 2.94e−017.52e−01 * * * * * * Test Hardy cases vs 0.002 (HWE) 0.015 (HWE) −0.011(HWE) −0.031 (HWE)   0.002 (HWE) 0.037 (HWE) Weinberg controls 0.764(HWD) 0.010 (HWE) −0.014 (HWE) −0.004 (HWE) −0.016 (HWE) 0.001 (HWE)

[0771] TABLE 20B HAPLOTYPE FREQUENCY ANALYSIS (Zyflo secondary effects)CASES (85 ALT+) vs CONTROLS (208 ALT−) ESTIMATED FREQUENCIESPERMUTATIONS Haplotype TEST RESULTS frequencies p- Odds Av. Max >Iter/MARKER 1 MARKER 2 MARKER 3 MARKER 4 MARKER 5 HAPLOTYPE cases controlsexcess ratio Chi-S P value (1 df) Chi-S Chi-s No. of Iter haplotype 1PT2 79 vs 192 12-197/244 12-196/119 TT 0.542 0.436 18.89 1.53 5.11(2.3e−02) ** 1.7 6.7 [3/100] haplotype 2 PT2 87 vs 197 12-208/3512-226/167 AG 0.123 0.070 5.73 1.87 4.34 (3.6e−02) * 1.1 7.3 [4/100]haplotype 3 PT2 84 vs 183 12-206/366 12-196/119 CC 0.205 0.139 7.68 1.603.75 (5.1e−02) * 1.3 8.0 [11/100]  haplotype 4 PT2 84 vs 192 10-347/22012-196/119 GT 0.486 0.400 14.25 1.41 3.49 (6.1e−02) * 1.0 5.3 [8/100]haplotype 5 PT2 81 vs 193 10-347/203 12-196/119 GC 0.207 0.144 7.46 1.563.43 (6.1e−02) * 1.3 10.3 [9/100] haplotype 6 PT3 82 vs 194 10-349/22412-216/421 12-223/207 TAT 0.158 0.064 10.09 2.76 12.35 (4.3e−04) *** 2.111.0 [0/100] haplotype 7 PT3 85 vs 186 12-206/366 12-216/421 12-223/207CAT 0.148 0.059 9.44 2.77 11.62 (6.3e−04) *** 2.8 24.7 [3/100] haplotype8 PT3 77 vs 180 12-197/244 12-206/366 12-196/119 TTT 0.434 0.286 20.671.91 10.62 (1.1e−03) *** 1.8 10.9 [1/100] haplotype 9 PT3 78 vs 19012-197/244 10-347/220 12-196/119 TGT 0.433 0.291 19.96 1.86 9.98(1.6e−03) *** 1.7 9.3 [0/100] haplotype 10 PT3 76 vs 187 12-197/24410-349/224 12-196/119 TGT 0.435 0.293 20.17 1.86 9.88 (1.7e−03) *** 1.86.9 [0/100] haplotype 11 PT3 77 vs 191 12-197/244 10-349/224 12-216/421CTA 0.137 0.056 8.56 2.66 9.76 (1.7e−03) *** 1.6 11.3 [1/100] haplotype12 PT3 75 vs 191 12-197/244 10-347/203 12-196/119 TAT 0.431 0.294 19.421.82 9.13 (2.4e−03) *** 1.3 6.1 [0/100] haplotype 13 PT4 81 vs 18312-206/366 10-349/224 12-216/421 12-223/207 CTAT 0.160 0.058 10.82 3.1014.38 (1.5e−04) **** 2.0 13.9 [0/100] haplotype 14 PT4 84 vs 18512-206/366 10-346/141 12-216/421 12-223/207 CGAT 0.158 0.058 10.62 3.0414.20 (1.6e−04) **** 3.3 23.7 [2/100] haplotype 15 PT4 82 vs 18810-349/224 12-214/129 12-216/421 12-223/207 TCAT 0.161 0.063 10.50 2.8613.12 (2.8e−04) **** 2.9 25.2 [3/100] haplotype 16 PT4 81 vs 18412-206/366 10-347/203 12-216/421 12-223/207 CGAT 0.153 0.059 10.03 2.8912.50 (3.9e−04) *** 2.7 13.0 [1/100] haplotype 17 PT4 82 vs 19110-347/111 10-349/224 12-216/421 12-223/207 GTAT 0.159 0.064 10.14 2.7712.33 (4.3e−04) *** 2.3 15.7 [2/100] haplotype 18 PT4 82 vs 19210-346/141 10-349/224 12-216/421 12-223/207 GTAT 0.158 0.065 9.96 2.7011.85 (5.6e−04) *** 2.6 18.1 [3/100] haplotype 19 PT4 81 vs 19210-347/220 10-349/224 12-216/421 12-223/207 ATAT 0.159 0.066 10.02 2.6911.78 (5.9e−04) *** 2.2 9.9 [0/100] haplotype 20 PT4 84 vs 18312-206/366 10-347/220 12-216/421 12-223/207 CAAT 0.150 0.059 9.60 2.7911.72 (5.9e−04) *** 2.3 17.1 [3/100] haplotype 21 PT4 85 vs 18312-206/366 10-347/111 12-216/421 12-223/207 CGAT 0.148 0.059 9.42 2.7611.45 (7.0e−04) *** 2.0 12.3 [1/100] haplotype 22 PT4 76 vs 18512-197/244 10-346/141 10-349/224 12-196/119 TGGT 0.435 0.284 21.12 1.9411.16 (8.2e−04) *** 2.0 8.7 [0/100] haplotype 23 PT4 85 vs 18012-206/366 12-214/129 12-216/421 12-223/207 CCAT 0.145 0.058 9.20 2.7411.03 (8.6e−04) *** 2.3 15.7 [3/100] haplotype 24 PT4 80 vs 19210-347/203 10-349/224 12-216/421 12-223/207 GTAT 0.156 0.065 9.65 2.6311.00 (8.6e−04) *** 1.7 14.4 [3/100] haplotype 25 PT4 82 vs 19010-347/203 10-341/116 12-214/129 12-223/207 GGCT 0.125 0.046 8.22 2.9410.86 (9.6e−04) *** 1.8 18.8 [2/100] haplotype 26 PT5 77 vs 19012-197/244 12-208/35 12-196/119 12-216/421 12-219/230 TATGA 0.138 0.0509.27 3.06 12.24 (4.5e−04) *** 2.6 13.2 [2/100] haplotype 27 PT5 77 vs189 12-197/244 12-208/35 10-349/97 12-196/119 12-223/207 TTATC 0.1270.045 8.56 3.08 11.42 (7.0e−04) *** 1.6 10.3 [0/100] haplotype 28 PT5 77vs 184 12-197/244 12-208/35 12-196/119 12-214/129 12-219/230 TATTA 0.1260.047 8.30 2.93 10.42 (1.2e−03) *** 2.4 14.6 [4/100] haplotype 29 PT5 76vs 188 12-197/244 12-208/35 10-347/220 12-196/119 12-223/207 TTGTC 0.1210.048 7.63 2.71 8.84 (2.9e−03) *** 1.4 9.5 [1/100] haplotype 30 PT5 76vs 176 12-197/244 12-208/35 12-206/366 10-341/116 12-196/119 TTTGT 0.1950.099 10.67 2.21 8.82 (2.9e−03) *** 1.3 7.3 [0/100]

[0772] TABLE 21 Summary of Association Study Results and PermutationTests 12-Lipoxygenase 12-206-366 10-347-203 10-349-224 12-196-11912-216-421 12-219-230 12-223-207 intron 2 exon 6 exon 8 MARKERS C T A THAPLOTYPE 8 Zyflo secondary effects (ALT+ vs ALT−) A T A G HAPLOTYPE 14Asthma (ALT vs US) 4.03E−01 5.84E−01 7.52E−01 4.03E−01 2.73E−01 2.94E−017.52E−01 p value ALT+ vs −3.7 −2.2 −1.4   3.4 −4.7   4.5 −0.6 diff all.Freq ALT− 2.73E−01 6.55E−01 5.27E−01 6.55E−01 3.71E−01 1.80E−01 7.52E−01p value ALT vs −4.6 −1.2 −1.9 −1.2 −2.9 −4.2   0.8 diff all. Freqcaucasian US sample sizes haplotype PERMUTATIONS HAPLOTYPE 8 (ALT+ vsALT−) Zyflo cases vs frequencies TEST RESULTS secondary effects controlscases controls odds-ratio chi-S P value Av. Chi-S Max Chi-S >Iter/nb ofIter. ALT+ vs ALT−  82 vs 174 0.158 0.04 4.56 21.85 2.90E−06 3.1 29.95/1 000 3.3 40.9 77/10 000 ALT vs caucasian US 256 vs 83  0.059 0  10.12 1.40E−03 3.5 37.6 82/1 000 sample sizes haplotype PERMUTATIONScases vs frequencies TEST RESULTS HAPLOTYPE 14 (ALT vs US) Asthmacontrols cases controls odds-ratio chi-S P value Av. Chi-S MaxChi-S >Iter/nb of Iter. ALT+ vs ALT−  85 vs 193 0.097 0.109 −1.34  0.186.50E−01 2.1 24.1 785/1 000 ALT vs caucasian US 278 vs 174 0.118 0.031  4.18 21.01 4.40E−06 2.8 38.6 39/10 000 2.8 29.9 7/1 000

[0773] TABLE 22 Permutations Test Results 12-Lipoxygenase 12-206/10-349/ 10-349/ 12-196/ 12-214/ 12-216/ 12-219/ 12-223/ 366 97 224 119129 421 230 207 in2 ex8 ex8 in bac (not localization in Bac: 3′ or 5′gene) MARKERS C T A T HAPLOTYPE 1 (ALT+ vs ALT−) A C G HAPLOTYPE 2 (ALTvs US) A T A G HAPLOTYPE 3 4.03e−01 7.52e−01 4.80e−01 4.03e−01 5.84e−012.73e−01 2.94e−01 7.52e−01 p value ALT+ vs −3.7 −0.1 −3.0   3.4 −2.4−4.7   4.5 −0.6 (cases vs ALT− (54 vs 58) (58 vs 59) (54 vs 57) (72 vs68) (58 vs 61) (57 vs 62) (66 vs 62) (61 vs 62) controls) 2.73e−016.55e−01 2.06e−01 6.55e−01 7.52e−01 3.71e−01 1.80e−01 7.52e−01 p valueALT vs −4.6 −1.4 −4.1 −1.2 −0.7 −2.9 −4.2   0.8 (cases vs caucasian US(57 vs 61) (59 vs 60) (56 vs 60) (69 vs 70) (60 vs 61) (61 vs 64) (63 vs67) (62 vs 61) controls) Zyflo secondary effects PERMUTATIONS samplesizes haplotype TEST RESULTS HAPLOTYPE 1 (ALT+ vs ALT−) cases vsfrequencies p- odds- Max (Zyflo secondary effects) CTAT controls casescontrols excess ratio chi-S P value Av Chi-S Chi-S >Iter/nb of Iter.ALT+ vs ALT−  81 vs 183 0.16 0.058 10.82 3.10 14.38 1.50E−04 **** 2 13.90/100 2.7 33.6 18/1000 ALT+ vs ALT− (1) 81 vs 99 0.16 0.065 10.11 2.728.28 4.00E−03 *** 3.3 23.1 118/1000 ALT+ vs ALT− (2) 81 vs 84 0.16 0.04412.12 4.15 12.23 4.50E−04 *** 2.7 19.6 20/1000 ALT vs caucasian US 264vs 83  0.071 0 302,77# 7.08 12.37 4.30E−04 ***TH 2.6 23 25/1000 Asthmagene sample sizes haplotype PERMUTATIONS HAPLOTYPE 2 (ALT vs US) casesvs frequencies p- odds- TEST RESULTS (Asthma gene) ACG controls casescontrols excess ratio chi-S P value Av. Chi-S Max Chi-S >Iter/nb ofIter. ALT+ vs ALT−  89 vs 193 0.131 0.121 1.14 1.10 0.11 6.50E−01 * 1.318.1 760/1000 ALT+ vs ALT− (1)  89 vs 104 0.131 0.115 1.84 1.16 0.205.80E−01 * 1.5 14.6 683/1000 ALT+ vs ALT− (2) 89 vs 89 0.131 0.134 −0.260.98 0.00 7.50E−01 * 1.4 16.2 946/1000 ALT vs caucasian US 282 vs 1740.125 0.041 8.73 3.32 17.87 2.30E−05 ***** 1.8 8.5 0/100 2 19.9 2/1000sample sizes haplotype PERMUTATIONS HAPLOTYPE 3 (ALT vs US) cases vsfrequencies p- odds- TEST RESULTS (Asthma gene) ATAG controls casescontrols excess ratio chi-S P value Av. Chi-S Max Chi-S >Iter/nb ofIter. ALT+ vs ALT−  86 vs 194 0.123 0.144 1.04 1.09 0.10 7.50E−01 * 1.515.5 816/1000 ALT+ vs ALT− (1)  86 vs 100 0.123 0.108 1.69 1.16 0.215.80E−01 * 1.6 16.6 735/1000 ALT+ vs ALT− (2) 86 vs 94 0.123 0.11 1.541.14 0.16 6.50E−01 * 1.5 19.3 750/1000 ALT vs caucasian US 280 vs 1730.124 0.035 9.2 3.9 20.63 5.40E−06 ****** 1.5 11.6 0/100 2 18.7 0/1000

[0774] TABLE 23 Allele Frequency ALT+ ALT− US Caucasian PROTEINS Markersize A C G T size A C G T size A C G T 12-LO 12-197/244 81 29.63 70.37196 35.46 64.54 180 32.78 67.22 2 12-208/35  87 41.95 58.05 197 41.1258.88 182 42.31 57.69 3 12-226/167 89 60.67 39.33 199 62.81 37.19 18858.78 41.22 4 12-206/366 86 45.35 54.65 186 41.67 58.33 89 38.20 61.80 510-346/141 88 HOM 197 0.51 99.49 185 HOM 6 10-347/111 88 0.57 99.43 196HOM 180 HOM 7 10-347/165 69 HOM 199 99.75 0.25 185 HOM 8 10-347/203 8356.02 43.98 197 57.87 42.13 184 58.42 41.58 9 10-347/220 87 42.53 57.47196 42.60 57.40 184 40.49 59.51 10 10-349/97  89 58.99 41.01 198 59.0940.91 182 60.44 39.56 11 10-349/224 83 54.22 45.78 194 57.22 42.78 17760.45 39.55 12 10-341/116 89 9.55 90.45 197 11.17 88.83 176 10.80 89.2013 12-196/119 86 27.91 72.09 195 31.28 68.72 184 29.08 70.92 1412-214/129 89 41.01 58.99 193 38.60 61.40 181 38.67 61.33 15 12-216/42189 42.13 57.87 199 37.44 62.56 182 35.99 64.01 16 12-219/230 89 66.8533.15 199 62.31 37.69 187 67.91 32.09 17 12-223/207 88 38.07 61.93 19937.44 62.56 186 38.44 61.56

[0775]

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030228582). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A method of determining the frequency in a population of an allele ofan eicosanoid-related biallelic marker or 12-LO-related biallelicmarker, comprising the steps of: (a) genotyping individuals from saidpopulation for said eicosanoid-related biallelic marker or 12-LO-relatedbiallelic marker according to the method of claim 1; and (b) determiningthe proportional representation of said eicosanoid-related biallelicmarker or 12-LO-related biallelic marker in said population.
 2. Themethod according to claim 1, wherein said eicosanoid-related biallelicmarker is found in a sequence selected from the group consisting of SEQID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304.
 3. The method according to claim 1, wherein step (a) isperformed on each individual of said population.
 4. The method accordingto claim 1, wherein step (a) is performed on a single pooled biologicalsample derived from said population.
 5. A method of detecting anassociation between an allele and a phenotype, comprising the steps of:(a) determining the frequency of at least one eicosanoid-relatedbiallelic marker allele or at least one 12-LO-related biallelic markerallele in an affected population according to the method of claim 1; (b)determining the frequency of said eicosanoid-related biallelic markerallele or said 12-LO-related biallelic marker allele in a controlpopulation according to the method of claim 1; and (c) determiningwhether a statistically significant association exists between saideicosanoid-related biallelic marker allele or said 12-LO-relatedbiallelic marker allele and said phenotype.
 6. A method of estimatingthe frequency of a haplotype for a set of biallelic markers in apopulation, comprising the steps of: (a) genotyping each individual insaid population for a first biallelic marker, wherein said firstbiallelic marker is a eicosanoid-related biallelic marker or a12-LO-related biallelic marker; (b) genotyping each individual in saidpopulation for a second biallelic marker by determining the identity ofthe nucleotides at said second biallelic marker for both copies of saidsecond biallelic marker present in the genome; and (c) applying ahaplotype determination method to the identities of the nucleotidesdetermined in steps (a) and (b) to obtain an estimate of said frequency.7. The method according to claim 6, wherein said haplotype determinationmethod is selected from the group consisting of asymmetric PCRamplification, double PCR amplification of specific alleles, the Clarkmethod, and an expectation maximization algorithm.
 8. The methodaccording to claim 5, wherein said eicosanoid-related biallelic markeris found in a sequence selected from the group consisting of SEQ ID Nos.651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304.
 9. A method of detecting an association between a haplotypeand a phenotype, comprising the steps of: (a) estimating the frequencyof at least one haplotype in an affected population according to themethod of claim 6; (b) estimating the frequency of said haplotype in acontrol population according to the method of claim 6; and (c)determining whether a statistically significant association existsbetween said haplotype and said phenotype.
 10. The method according toclaim 5, wherein said control population is either a trait negativepopulation or a random population.
 11. The method according to claim 5,wherein steps (a) and (b) are performed on a single pooled biologicalsample derived from each of said populations.
 12. The method accordingto claim 5, wherein steps (a) and (b) are performed separately onbiological samples derived from each individual in said populations. 13.The method according to claim 5, wherein said phenotype is a diseaseinvolving arachidonic acid metabolism.
 14. The method according to claim5, wherein said phenotype is a response to an agent acting onarachidonic acid metabolism.
 15. The method according to claim 5,wherein said phenotype is a side effect to an agent acting onarachidonic acid metabolism.
 16. The method according to claim 5,wherein the identity of the nucleotides at all of the biallelic markersdescribed in FIG. 2(A-B) is determined in steps (a) and (b).
 17. Themethod according to claim 6, wherein said eicosanoid-related biallelicmarker is found in a sequence selected from the group consisting of SEQID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and1195-1304.
 18. The method according to claim 9, wherein said controlpopulation is either a trait negative population or a random population.19. The method according to claim 9, wherein said phenotype is a diseaseinvolving arachidonic acid metabolism.
 20. The method according to claim9, wherein said phenotype is a response to an agent acting onarachidonic acid metabolism.
 21. The method according to claim 9,wherein said phenotype is a side effect to an agent acting onarachidonic acid metabolism.